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  • 99
    Millipore human serum albumin hsa
    The 3 - D fluorescence spectra of <t>BSA</t> and <t>HSA.</t> A. The 3-D fluorescence spectra and corresponding contour diagrams of BSA (A) and BSA-HU (B). The concentration of protein was 5 μM and that of HU was 20 μM. B. The 3-D fluorescence spectra and corresponding contour diagrams of HSA (A) and HSA-HU (B) . The concentration of protein was 5 μM and that of HU was 20 μM.
    Human Serum Albumin Hsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 398 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher exosome depleted fetal bovine serum
    Physical analysis of nanovesicles isolated from brains of P301L tau transgenic rTg4510 mice. <t>Exosome-like</t> EVs were isolated from the brain extracellular space according to Perez-Gonzalez et al. ( 20 ). A , transmission electron microscopy analysis of sucrose F1-F6 shows nanovesicles mostly in F2 (0.6 m sucrose, ρ = 1.08g/ml) and F3 (0.95 m sucrose, ρ = 1.12g/ml). Scale bars = 100 nm. B , tunable resistive pulse sensing using a qNano instrument (Izon). Concentration histograms of size distributions (sizes in nanometers versus particles per milliliter) are shown for each population in the different sucrose fractions (F1-F6). In agreement with transmission electron microscopy, the highest concentrations of nanovesicles were found in F2 (0.6 m ) and F3 (0.95 m ). The size distribution is compatible with exosomes for both WT and rTg4510 Tg nanovesicles, with an average size of 130 nm in F3 (0.95 m sucrose) and the most common nanovesicle size of ∼74 nm for both WT and Tg samples. However, some larger extracellular vesicles ( > 130 nm) were co-isolated with these potential exosomes. More exosome-like EVs were obtained from the WT samples than the Tg samples.
    Exosome Depleted Fetal Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher keratinocyte serum free medium
    Transient expression of OCT4 produces a change in methylation state of the endogenous OCT4 and NANOG promoters in the transfected human skin <t>keratinocytes</t> (HSK-OCT4-GFP). Shown are methylation states of CpG islands −929 to +421 around the transcription start site of the endogenous OCT4 promoter and upstream areas −1091 to −299 of the NANOG promoter for the NTERA-2 cell line (positive control), human skin keratinocytes (HSK-GFP) 48 hours after transient transfection with pcDNA3-GFP (negative control), and human skin keratinocytes (HSK-OCT4-GFP) 48 hours after transient transfection with pcDNA3-OCT4-GFP. Transfected keratinocytes were sorted from untransfected keratinocytes based on GFP; thus the analysis was done only on the transfected cells. Note, demethylation of OCT4 was consistent at +74 and +80, and demethylation of NANOG was consistent at −649, −611, −565, −516, and −378. Numbers relative to translational start site.
    Keratinocyte Serum Free Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    STEMCELL Technologies Inc stemspan serum free medium
    The effects of collagen I gel concentrations on CD133 + cell migration in the 3D μ-slide assay. (A) After 24 hour culture of human UCB CD133 + cells in <t>StemSpan</t> medium and SCF, Flt-3 ligand, IL-6 and TPO, cells were suspended in 0.5 mg/ml, 1 mg/ml or 3 mg/ml collagen I gel solutions and seeded into the central chamber of the 3D chemotaxis μ-slides. A chemokine gradient was established using 1 μg/ml CXCL12. The cells were imaged over 22 h by timelapse microscopy migration and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug in for (a) accumulated distance, (b) velocity, (c) displacement of the center of mass and (d) forward migration index. Values are means ± SEM (n ≥ 3independent experiments). Variability amongst independent experiments was increased when 3 mg/ml collagen I gels were compared with 0.5 or 1 mg/ml collagen I gels. This is exemplified when comparing 1 mg/ml and 3 mg/ml collagen I gels respectively for the ranges in the accumulated distance (1012 μm to 1451 μm vs. 350 μm to 1467 μm respectively), velocity (0.77 to 1.16 vs. 0.27 to 1.18 μm/min respectively), displacement of center of mass (111.80 to 115.43 vs. 26.94 to 256.20 μm respectively) and forward migration index (0.100 to 0.110 vs. 0.060 to 0.220 respectively). (B) Microscopic images (× 4 magnification) of CD133 + cells in (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 3 mg/ml collagen 1 gels after seeding into the central chamber of the 3D chemotaxis μ-slide and demonstrating the lack of uniformity of collagen I fibers with 3 mg/ml collagen I gels.
    Stemspan Serum Free Medium, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 99/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bovine serum albumin bsa
    The 3 - D fluorescence spectra of <t>BSA</t> and <t>HSA.</t> A. The 3-D fluorescence spectra and corresponding contour diagrams of BSA (A) and BSA-HU (B). The concentration of protein was 5 μM and that of HU was 20 μM. B. The 3-D fluorescence spectra and corresponding contour diagrams of HSA (A) and HSA-HU (B) . The concentration of protein was 5 μM and that of HU was 20 μM.
    Bovine Serum Albumin Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13463 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare fetal bovine serum fbs
    Effects of cycloheximide on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 10 μM cycloheximide for 24 hours in <t>DMEM</t> containing 10% <t>FBS</t> without (Chx) or with 33% vitreous humor (V/Chx). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P
    Fetal Bovine Serum Fbs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 11587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    FUJIFILM bovine serum albumin bsa
    Effect of FGF2 and/or TGFβ2 stimulation on activation of ERK1/2 pathway in MLECs. To evaluate the effect of FGF2 with/without TGFβ2 stimulation on phosphorylation of ERK1/2 in MLECs, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in <t>DMEM</t> containing 0.1% <t>BSA</t> for 10 or 60 min. Cell lysates were prepared, and Western blotting analysis was performed using anti‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab or anti‐phospho‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab. Data were from three experiments and were reported as means ± S.D.s.
    Bovine Serum Albumin Bsa, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 96/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Quidel c1q depleted human serum
    CHX partially inhibited complement-mediated ApoE accumulation. RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant ( A ) and a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant ( B ) were preincubated with CHX at 20 μg/mL for 1.5 hours, primed with or without S58 (1.2 mg/mL) for 30 minutes, and then treated with either 6% <t>C1q-Dep</t> or HiC1q-Dep for 5 hours. CHX was continuously present when cells were primed with S58 and incubated with complement serum. Total proteins (15 μg) were separated by SDS-PAGE for Western blot. The quantity of ApoE relative to GAPDH shown in ( A , B ) was determined by densitometry (labeling 1–5 represents five treatment groups). * P
    C1q Depleted Human Serum, supplied by Quidel, used in various techniques. Bioz Stars score: 89/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher serum free keratinocyte media
    HPV18 requires EGFR activation to maintain cyclin B1 expression in differentiating cells (A) Schematic showing the EGFR/ERK signalling pathway and the targets of the siRNA and inhibitors used in this study. Mock treatedkeratinocytes were differentiated in high calcium media and lysed after 48 hours. In parallel, <t>keratinocytes</t> were treated with (B) scramble or EGFR specific siRNA, (C) an EGFR kinase inhibitor (2 μM PD153035), or (D) a Mek1/2 kinase inhibitor (20 μM UO126) during differentiation. All samples were analysed for cyclin B1 and phosphorylated ERK1/2 expression. GAPDH served as a loading control. Representative blots are shown from at least three independent biological repeats from two donor lines.
    Serum Free Keratinocyte Media, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    STEMCELL Technologies Inc serum free stemspan media
    The effects of collagen I gel concentrations on CD133 + cell migration in the 3D μ-slide assay. (A) After 24 hour culture of human UCB CD133 + cells in <t>StemSpan</t> medium and SCF, Flt-3 ligand, IL-6 and TPO, cells were suspended in 0.5 mg/ml, 1 mg/ml or 3 mg/ml collagen I gel solutions and seeded into the central chamber of the 3D chemotaxis μ-slides. A chemokine gradient was established using 1 μg/ml CXCL12. The cells were imaged over 22 h by timelapse microscopy migration and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug in for (a) accumulated distance, (b) velocity, (c) displacement of the center of mass and (d) forward migration index. Values are means ± SEM (n ≥ 3independent experiments). Variability amongst independent experiments was increased when 3 mg/ml collagen I gels were compared with 0.5 or 1 mg/ml collagen I gels. This is exemplified when comparing 1 mg/ml and 3 mg/ml collagen I gels respectively for the ranges in the accumulated distance (1012 μm to 1451 μm vs. 350 μm to 1467 μm respectively), velocity (0.77 to 1.16 vs. 0.27 to 1.18 μm/min respectively), displacement of center of mass (111.80 to 115.43 vs. 26.94 to 256.20 μm respectively) and forward migration index (0.100 to 0.110 vs. 0.060 to 0.220 respectively). (B) Microscopic images (× 4 magnification) of CD133 + cells in (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 3 mg/ml collagen 1 gels after seeding into the central chamber of the 3D chemotaxis μ-slide and demonstrating the lack of uniformity of collagen I fibers with 3 mg/ml collagen I gels.
    Serum Free Stemspan Media, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 85/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Abcam iglon5 positive human serum samples
    IgG1 <t>IgLON5</t> antibodies internalize IgLON5 clusters. Hippocampal neurons treated for 3 days with total IgG, IgG1, and IgG4 IgLON5 antibodies. The immunofluorescence strategy to differentiate surface and internal human IgG bound to IgLON5 is conducted as in Fig. 8 . The IgG1 antibodies alone could reproduce the same effects seen with the total IgG; meanwhile, the IgG4 did not produce internalization. Scale bar = 5 μm
    Iglon5 Positive Human Serum Samples, supplied by Abcam, used in various techniques. Bioz Stars score: 84/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    GeneTex anti ie2 serum
    <t>IE2</t> is degraded in the absence of its E3 ligase activity. Wild type (WT) IE2 and IE2C230S (containing a RING domain mutation)-expressing recombinant viruses were transduced into Vero E6 cells together with KNK437 treatment at various concentrations. The cell extracts were collected at 48 hpt for the detection of IE2 protein levels.
    Anti Ie2 Serum, supplied by GeneTex, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Cambrex serum free keratinocyte growth medium
    The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human <t>keratinocytes</t>
    Serum Free Keratinocyte Growth Medium, supplied by Cambrex, used in various techniques. Bioz Stars score: 82/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher serum teir
    Expression of miR-16 and miR-451 in the exosomes isolated from the different techniques and volumes of serum. ddPCR was used to measure the absolute concentration of miR-16 and miR-451 in the exosomes isolated with miRCURY (blue), <t>ExoQuick</t> (green), <t>TEIR</t> (red), and UC (yellow) using the different volumes of human serum. The values in the graph are the mean concentration (copies/μl) ± SEM (n = 3), *p≤0.05.
    Serum Teir, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Santa Cruz Biotechnology goat serum anti cyclin d3
    Binding of <t>cyclin</t> D3 to pRb in vitro. The schematic in the upper inset shows the structure of the GST-Rb fusion proteins used and illustrates the A/B pocket region and the carboxy-terminal region of Rb. The lower inset shows schematic representations
    Goat Serum Anti Cyclin D3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Abcam anti riz1 rabbit serum
    ChIP analysis of estrogen target genes. (A) Soluble chromatin was prepared from MCF7 cells not treated or treated with E2 for 45 min. Immunoprecipitation was performed with antibodies against <t>RIZ1,</t> dimethylated H3-K9, dimethylated H3-K4, acetylated H3-K9,
    Anti Riz1 Rabbit Serum, supplied by Abcam, used in various techniques. Bioz Stars score: 78/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Wyeth Biopharma sheep anti cho hcp serum
    Isotyping of the <t>Anti-CHO-HCP</t> Antibodies in Normal Human Serum Samples
    Sheep Anti Cho Hcp Serum, supplied by Wyeth Biopharma, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The 3 - D fluorescence spectra of BSA and HSA. A. The 3-D fluorescence spectra and corresponding contour diagrams of BSA (A) and BSA-HU (B). The concentration of protein was 5 μM and that of HU was 20 μM. B. The 3-D fluorescence spectra and corresponding contour diagrams of HSA (A) and HSA-HU (B) . The concentration of protein was 5 μM and that of HU was 20 μM.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: The 3 - D fluorescence spectra of BSA and HSA. A. The 3-D fluorescence spectra and corresponding contour diagrams of BSA (A) and BSA-HU (B). The concentration of protein was 5 μM and that of HU was 20 μM. B. The 3-D fluorescence spectra and corresponding contour diagrams of HSA (A) and HSA-HU (B) . The concentration of protein was 5 μM and that of HU was 20 μM.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Fluorescence, Concentration Assay

    FT- IR spectra of (A) BSA- HU system and (B) HSA- HU system.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: FT- IR spectra of (A) BSA- HU system and (B) HSA- HU system.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques:

    Synchronous fluorescence spectra of BSA and HSA. A. Synchronous fluorescence spectra of BSA-HU: For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 μM. The concentration of BSA was 5.0 μM. B. Synchronous fluorescence spectra of HSA-HU: (A) For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20, (f) 25 and (g) 30 μM. The concentration of HSA was 5.0 μM.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Synchronous fluorescence spectra of BSA and HSA. A. Synchronous fluorescence spectra of BSA-HU: For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 μM. The concentration of BSA was 5.0 μM. B. Synchronous fluorescence spectra of HSA-HU: (A) For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20, (f) 25 and (g) 30 μM. The concentration of HSA was 5.0 μM.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Fluorescence, Concentration Assay

    Absorbance spectra of BSA and HSA. A. Absorbance spectra of BSA, HU and HU-BSA system. BSA concentration was 5 μM (a). HU concentration for HU–BSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU. B. Absorbance spectra of HSA, HU and HU-HSA system. HSA concentration was 5 μM (a). HU concentration for HU–HSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Absorbance spectra of BSA and HSA. A. Absorbance spectra of BSA, HU and HU-BSA system. BSA concentration was 5 μM (a). HU concentration for HU–BSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU. B. Absorbance spectra of HSA, HU and HU-HSA system. HSA concentration was 5 μM (a). HU concentration for HU–HSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Concentration Assay

    Structure of BSA and HSA.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Structure of BSA and HSA.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques:

    Van’t Hoff plot with BSA and HSA. A. van’t Hoff plot for the binding of HU with BSA. B. van’t Hoff plot for the binding of HU with HSA.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Van’t Hoff plot with BSA and HSA. A. van’t Hoff plot for the binding of HU with BSA. B. van’t Hoff plot for the binding of HU with HSA.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Binding Assay

    Physical analysis of nanovesicles isolated from brains of P301L tau transgenic rTg4510 mice. Exosome-like EVs were isolated from the brain extracellular space according to Perez-Gonzalez et al. ( 20 ). A , transmission electron microscopy analysis of sucrose F1-F6 shows nanovesicles mostly in F2 (0.6 m sucrose, ρ = 1.08g/ml) and F3 (0.95 m sucrose, ρ = 1.12g/ml). Scale bars = 100 nm. B , tunable resistive pulse sensing using a qNano instrument (Izon). Concentration histograms of size distributions (sizes in nanometers versus particles per milliliter) are shown for each population in the different sucrose fractions (F1-F6). In agreement with transmission electron microscopy, the highest concentrations of nanovesicles were found in F2 (0.6 m ) and F3 (0.95 m ). The size distribution is compatible with exosomes for both WT and rTg4510 Tg nanovesicles, with an average size of 130 nm in F3 (0.95 m sucrose) and the most common nanovesicle size of ∼74 nm for both WT and Tg samples. However, some larger extracellular vesicles ( > 130 nm) were co-isolated with these potential exosomes. More exosome-like EVs were obtained from the WT samples than the Tg samples.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Physical analysis of nanovesicles isolated from brains of P301L tau transgenic rTg4510 mice. Exosome-like EVs were isolated from the brain extracellular space according to Perez-Gonzalez et al. ( 20 ). A , transmission electron microscopy analysis of sucrose F1-F6 shows nanovesicles mostly in F2 (0.6 m sucrose, ρ = 1.08g/ml) and F3 (0.95 m sucrose, ρ = 1.12g/ml). Scale bars = 100 nm. B , tunable resistive pulse sensing using a qNano instrument (Izon). Concentration histograms of size distributions (sizes in nanometers versus particles per milliliter) are shown for each population in the different sucrose fractions (F1-F6). In agreement with transmission electron microscopy, the highest concentrations of nanovesicles were found in F2 (0.6 m ) and F3 (0.95 m ). The size distribution is compatible with exosomes for both WT and rTg4510 Tg nanovesicles, with an average size of 130 nm in F3 (0.95 m sucrose) and the most common nanovesicle size of ∼74 nm for both WT and Tg samples. However, some larger extracellular vesicles ( > 130 nm) were co-isolated with these potential exosomes. More exosome-like EVs were obtained from the WT samples than the Tg samples.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Isolation, Transgenic Assay, Mouse Assay, Transmission Assay, Electron Microscopy, Tunable Resistive Pulse Sensing, Concentration Assay

    Exosome-like EVs can induce endogenous tau aggregation in FRET tau biosensor cells. FRET tau biosensor cells are HEK293T cells that express both tau-CFP and tau-YFP (RD domain). Cells were treated and analyzed by FRET flow cytometry after a 24-h treatment ( n = 3, average ± S.D., 20,000 cells/experiment were analyzed). Representative flow cytometry plots show the FRET signal as detected in the right gate (Q2, shaded in green ). A , titration of protein sample to determine concentrations near saturation of FRET tau biosensor cells. Cells were treated with decreasing amounts of rTg4510 transgenic brain cell lysates in the presence of Lipofectamine and analyzed by FRET flow cytometry after a 24-h treatment. 20 μg of protein in cell lysates is sufficient to generate a FRET signal that is not much lower than that obtained with only 1.0 or 0.5 μg of protein. Even with 8 ng of protein, a FRET signal is detected. Therefore, cells are saturated at 20 μg of cell lysate and detect tau seeds up to the low nanogram range. This implies that even minute amounts of tau seeds are detectable in 20 μg of protein. ex , excitation. B–J , analysis of the requirement of Lipofectamine. FRET tau biosensor cells were treated in the presence ( C , D , G , and H ) or absence ( E , F , I , and J ) of Lipofectamine, with vehicle ( B ) showing a lack of signal in the gate for FRET (upper right gate Q2, shaded green ). C–F , none of the treatments with WT EVs or cell lysates can induce FRET. G–J , a strong FRET signal is detected with Tg cell lysates ( G ) and Tg EVs ( H ), but only in the presence of Lipofectamine. I and J , the same Tg samples without using Lipofectamine.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Exosome-like EVs can induce endogenous tau aggregation in FRET tau biosensor cells. FRET tau biosensor cells are HEK293T cells that express both tau-CFP and tau-YFP (RD domain). Cells were treated and analyzed by FRET flow cytometry after a 24-h treatment ( n = 3, average ± S.D., 20,000 cells/experiment were analyzed). Representative flow cytometry plots show the FRET signal as detected in the right gate (Q2, shaded in green ). A , titration of protein sample to determine concentrations near saturation of FRET tau biosensor cells. Cells were treated with decreasing amounts of rTg4510 transgenic brain cell lysates in the presence of Lipofectamine and analyzed by FRET flow cytometry after a 24-h treatment. 20 μg of protein in cell lysates is sufficient to generate a FRET signal that is not much lower than that obtained with only 1.0 or 0.5 μg of protein. Even with 8 ng of protein, a FRET signal is detected. Therefore, cells are saturated at 20 μg of cell lysate and detect tau seeds up to the low nanogram range. This implies that even minute amounts of tau seeds are detectable in 20 μg of protein. ex , excitation. B–J , analysis of the requirement of Lipofectamine. FRET tau biosensor cells were treated in the presence ( C , D , G , and H ) or absence ( E , F , I , and J ) of Lipofectamine, with vehicle ( B ) showing a lack of signal in the gate for FRET (upper right gate Q2, shaded green ). C–F , none of the treatments with WT EVs or cell lysates can induce FRET. G–J , a strong FRET signal is detected with Tg cell lysates ( G ) and Tg EVs ( H ), but only in the presence of Lipofectamine. I and J , the same Tg samples without using Lipofectamine.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Flow Cytometry, Cytometry, Titration, Transgenic Assay

    Effect of an extended culture on the generation of the FRET signal and tau aggregation. FRET tau biosensor cells were treated in the presence ( B , C , F , and G ) or absence ( D , E , H , and I ) of Lipofectamine and analyzed by FRET flow cytometry after a 72-h treatment ( n = 3, average ± S.D.. 40,000 cells/experiment were analyzed). A , FRET tau biosensor cells treated only with Lipofectamine do not induce FRET (upper right gate Q2, shaded green ). ex , excitation. B–E , none of the treatments with WT exosome-like EVs or cell lysates triggers FRET. F and G , a strong FRET signal is detected with Tg cell lysates ( F ) and Tg exosome-like EVs ( G ) in the presence of Lipofectamine. H and I , when Lipofectamine is omitted, a weak signal is detected for Tg lysates ( H ) and also for Tg EVs ( I). J–R , parallel experiment using the same conditions was performed with cells grown on coverslips to then be analyzed by confocal microscopy. Scale bar = 50 μm. O–R , induced tau aggregates were visualized only with Tg samples. S , quantification of the ratio of the area occupied by tau inclusions normalized by the area occupied by nuclei 72-h after treatment (tau aggregates/DAPI in percent, n = 4).

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Effect of an extended culture on the generation of the FRET signal and tau aggregation. FRET tau biosensor cells were treated in the presence ( B , C , F , and G ) or absence ( D , E , H , and I ) of Lipofectamine and analyzed by FRET flow cytometry after a 72-h treatment ( n = 3, average ± S.D.. 40,000 cells/experiment were analyzed). A , FRET tau biosensor cells treated only with Lipofectamine do not induce FRET (upper right gate Q2, shaded green ). ex , excitation. B–E , none of the treatments with WT exosome-like EVs or cell lysates triggers FRET. F and G , a strong FRET signal is detected with Tg cell lysates ( F ) and Tg exosome-like EVs ( G ) in the presence of Lipofectamine. H and I , when Lipofectamine is omitted, a weak signal is detected for Tg lysates ( H ) and also for Tg EVs ( I). J–R , parallel experiment using the same conditions was performed with cells grown on coverslips to then be analyzed by confocal microscopy. Scale bar = 50 μm. O–R , induced tau aggregates were visualized only with Tg samples. S , quantification of the ratio of the area occupied by tau inclusions normalized by the area occupied by nuclei 72-h after treatment (tau aggregates/DAPI in percent, n = 4).

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Flow Cytometry, Cytometry, Confocal Microscopy

    Only cells harboring tau inclusions secrete exosome-like EVs carrying tau seeds. Tau biosensor cells were used as an in vitro cellular model to isolate exosomes from a cell line in which tau aggregation was induced. Scale bar = 50 μm ( A , B , F , and G ) and 100 nm ( C and D ). A , fluorescent live-cell imagining of tau biosensor cells treated with WT mouse brain cell lysates to generate cultures without tau aggregation. B , cultures of cells forming tau inclusions ( bright green aggregates ) were obtained by treating the tau biosensor cells with rTg4510 brain lysates. C and D , electron microscopy of exosome-like EVs isolated from CCM of WT ( C , Wt-EVs ) or Tg cell cultures ( D , Tg-EVs ), showing that both types of cultures generate mostly EVs of less than 100 nm in diameter. E , Western blotting analysis of isolated EVs. 5 μg of protein equivalents of sucrose-purified EVs (F3, 0.95 m sucrose) were blotted. 5 and 20 μg of protein of corresponding cell lysates are included for comparison. Using equivalent protein quantities, both EVs and cell lysates show strong up-regulation of the exosome markers ALIX and FLOT-1. The endoplasmic reticulum marker calnexin ( CANX ) was undetectable. Total tau was detected with the Tau-RD4 antibody, showing that tau is present at similar levels in both WT and Tg EVs. SDS-stable high molecular weight ( MW , asterisks ) tau species were only detected with 20 μg of transgenic cell lysates. Increased tau phosphorylation (Ser(P)-262) was detected in cells undergoing cellular tau aggregation. However, Ser(P)-262 was undetectable with the amount of EV protein analyzed. C.E , cell extract. F , exosome-like EVs from WT cultures (WT EVs + Lipofectamine) are unable to induce tau aggregation in naïve tau biosensor cells. G , fluorescent live-cell imagining of tau biosensor cells treated with Tg EVs + Lipofectamine, showing the robust presence of induced tau aggregates. H and I , FRET flow cytometry of treatments with 5 μg of protein equivalents of WT EVs ( H ) and Tg EVs ( I ) showing that only Tg EVs are able to generate FRET signals indicative of tau aggregation ( n = 3, average ± S.D., 40,000 cells analyzed per experiment). ex , excitation. J , ThT fluorescence assay of HEK293-derived EVs ( n = 3, average fluorescence ± S.E.; a.u. , arbitrary units). A comparatively stronger increase of ThT fluorescence in Tg EVs indicates a higher presence of misfolded and aggregated tau.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Only cells harboring tau inclusions secrete exosome-like EVs carrying tau seeds. Tau biosensor cells were used as an in vitro cellular model to isolate exosomes from a cell line in which tau aggregation was induced. Scale bar = 50 μm ( A , B , F , and G ) and 100 nm ( C and D ). A , fluorescent live-cell imagining of tau biosensor cells treated with WT mouse brain cell lysates to generate cultures without tau aggregation. B , cultures of cells forming tau inclusions ( bright green aggregates ) were obtained by treating the tau biosensor cells with rTg4510 brain lysates. C and D , electron microscopy of exosome-like EVs isolated from CCM of WT ( C , Wt-EVs ) or Tg cell cultures ( D , Tg-EVs ), showing that both types of cultures generate mostly EVs of less than 100 nm in diameter. E , Western blotting analysis of isolated EVs. 5 μg of protein equivalents of sucrose-purified EVs (F3, 0.95 m sucrose) were blotted. 5 and 20 μg of protein of corresponding cell lysates are included for comparison. Using equivalent protein quantities, both EVs and cell lysates show strong up-regulation of the exosome markers ALIX and FLOT-1. The endoplasmic reticulum marker calnexin ( CANX ) was undetectable. Total tau was detected with the Tau-RD4 antibody, showing that tau is present at similar levels in both WT and Tg EVs. SDS-stable high molecular weight ( MW , asterisks ) tau species were only detected with 20 μg of transgenic cell lysates. Increased tau phosphorylation (Ser(P)-262) was detected in cells undergoing cellular tau aggregation. However, Ser(P)-262 was undetectable with the amount of EV protein analyzed. C.E , cell extract. F , exosome-like EVs from WT cultures (WT EVs + Lipofectamine) are unable to induce tau aggregation in naïve tau biosensor cells. G , fluorescent live-cell imagining of tau biosensor cells treated with Tg EVs + Lipofectamine, showing the robust presence of induced tau aggregates. H and I , FRET flow cytometry of treatments with 5 μg of protein equivalents of WT EVs ( H ) and Tg EVs ( I ) showing that only Tg EVs are able to generate FRET signals indicative of tau aggregation ( n = 3, average ± S.D., 40,000 cells analyzed per experiment). ex , excitation. J , ThT fluorescence assay of HEK293-derived EVs ( n = 3, average fluorescence ± S.E.; a.u. , arbitrary units). A comparatively stronger increase of ThT fluorescence in Tg EVs indicates a higher presence of misfolded and aggregated tau.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: In Vitro, Electron Microscopy, Isolation, Western Blot, Purification, Marker, Molecular Weight, Transgenic Assay, Flow Cytometry, Cytometry, Fluorescence, Derivative Assay

    Lipofectamine increases the uptake of EVs and protein aggregates that trigger intracellular tau inclusions that are closely associated with internalized seeds. A–K , tau biosensor cells were treated in the presence ( B , C , F , G , J , and K ) or absence ( D , E , H , and I ) of Lipofectamine, analyzed by confocal microscopy after 24-h treatment. To visualize uptake by cells, cell lysates were labeled on the N terminus of proteins with AF647. Membranes of EVs were labeled with the lipophilic far-red dye Cell Vue Claret (CV) for a similar internalization assay. Endogenous tau was detected by green/yellow emission (530–600 nm). Scale bars = 50 μm ( A–K ) and 20 μm ( M and N ). A , FRET tau biosensor cells treated with Lipofectamine vehicle only. No far-red signal is detected because no exosome-like EVs or cell lysates were added. B–E , none of the treatments with labeled WT exosome-like EVs or WT cell lysates induces aggregation of endogenous tau. F–I , treatments with labeled Tg cell lysates or exosome-like EVs. Bright tau inclusions are visualized with AF647 Tg cell lysates ( F ) and CV-Claret Tg EVs ( G ) in the presence of Lipofectamine. Tau inclusions are also detected with AF647 Tg cell lysates ( H ) or CV-Claret Tg EVs ( I ) in the absence of Lipofectamine. J–K , Lipofectamine-mediated uptake of P301L Tg cell lysates and exosome-like EVs without far-red label, used as positive controls. L , quantification of the ratio of the areas of induced tau aggregates normalized over the area of nuclei detected 24 h after treatment for Tg samples shown in F–I (tau aggregates/DAPI in percent, n = 4). M and N , high-magnification images of the internalization of far-red labeled Tg cell lysates ( M ) and Tg exosomes ( N ) in the presence of Lipofectamine. M , tau inclusions form in close proximity to internalized exogenous protein aggregates, sometimes completely surrounding the seed. N , this is also observed for internalized exosome-like EVs, where membranes are still showing Cell Vue Claret stain, whereas EVs nucleate the formation of intracellular tau inclusions. The signal of Cell Vue Claret suggests that there is a compartmentalized membrane involved in the presentation of tau seed to the cell for triggering aggregation.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Lipofectamine increases the uptake of EVs and protein aggregates that trigger intracellular tau inclusions that are closely associated with internalized seeds. A–K , tau biosensor cells were treated in the presence ( B , C , F , G , J , and K ) or absence ( D , E , H , and I ) of Lipofectamine, analyzed by confocal microscopy after 24-h treatment. To visualize uptake by cells, cell lysates were labeled on the N terminus of proteins with AF647. Membranes of EVs were labeled with the lipophilic far-red dye Cell Vue Claret (CV) for a similar internalization assay. Endogenous tau was detected by green/yellow emission (530–600 nm). Scale bars = 50 μm ( A–K ) and 20 μm ( M and N ). A , FRET tau biosensor cells treated with Lipofectamine vehicle only. No far-red signal is detected because no exosome-like EVs or cell lysates were added. B–E , none of the treatments with labeled WT exosome-like EVs or WT cell lysates induces aggregation of endogenous tau. F–I , treatments with labeled Tg cell lysates or exosome-like EVs. Bright tau inclusions are visualized with AF647 Tg cell lysates ( F ) and CV-Claret Tg EVs ( G ) in the presence of Lipofectamine. Tau inclusions are also detected with AF647 Tg cell lysates ( H ) or CV-Claret Tg EVs ( I ) in the absence of Lipofectamine. J–K , Lipofectamine-mediated uptake of P301L Tg cell lysates and exosome-like EVs without far-red label, used as positive controls. L , quantification of the ratio of the areas of induced tau aggregates normalized over the area of nuclei detected 24 h after treatment for Tg samples shown in F–I (tau aggregates/DAPI in percent, n = 4). M and N , high-magnification images of the internalization of far-red labeled Tg cell lysates ( M ) and Tg exosomes ( N ) in the presence of Lipofectamine. M , tau inclusions form in close proximity to internalized exogenous protein aggregates, sometimes completely surrounding the seed. N , this is also observed for internalized exosome-like EVs, where membranes are still showing Cell Vue Claret stain, whereas EVs nucleate the formation of intracellular tau inclusions. The signal of Cell Vue Claret suggests that there is a compartmentalized membrane involved in the presentation of tau seed to the cell for triggering aggregation.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Confocal Microscopy, Labeling, Staining

    Alix and flotillin-1 are also immunocolocalized on EVs after cellular uptake. Shown is Lipofectamine-mediated internalization of mouse brain-derived EVs labeled with Cell Vue Claret by tau biosensor cells. The arrowheads indicate sites of Alix and flotillin-1 colocalization. The corresponding grayscale-inverted look-up table images are shown below each immunofluorescence image for better visualization ( E–H and M–P ). A–D , exosome-like EVs from WT mice ( Wt-EVs ) were robustly internalized but failed to engage in tau aggregation. However, ALIX, forming intracellular puncta, and FLOT-1 colocalized in internalized EVs ( arrowheads ). I–L , exosome-like EVs from rTg4510 transgenic mice ( Tg-EVs ) strongly induced intracellular tau inclusions ( bright green ) in recipient cells. In the middle of this reorganized cytoplasm, some colocalization of ALIX and FLOT-1 is observed, sometimes buried in EVs inside tau inclusions ( arrowheads ). Scale bar = 20 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Alix and flotillin-1 are also immunocolocalized on EVs after cellular uptake. Shown is Lipofectamine-mediated internalization of mouse brain-derived EVs labeled with Cell Vue Claret by tau biosensor cells. The arrowheads indicate sites of Alix and flotillin-1 colocalization. The corresponding grayscale-inverted look-up table images are shown below each immunofluorescence image for better visualization ( E–H and M–P ). A–D , exosome-like EVs from WT mice ( Wt-EVs ) were robustly internalized but failed to engage in tau aggregation. However, ALIX, forming intracellular puncta, and FLOT-1 colocalized in internalized EVs ( arrowheads ). I–L , exosome-like EVs from rTg4510 transgenic mice ( Tg-EVs ) strongly induced intracellular tau inclusions ( bright green ) in recipient cells. In the middle of this reorganized cytoplasm, some colocalization of ALIX and FLOT-1 is observed, sometimes buried in EVs inside tau inclusions ( arrowheads ). Scale bar = 20 μm.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Derivative Assay, Labeling, Immunofluorescence, Mouse Assay, Transgenic Assay

    Levels of uptake of exosome-like EVs and proteins in cell lysates determine the generation of the FRET signal. Tau biosensor cells were treated in the presence ( B , C , F , G , J , and K ) or absence ( D , E , H , and I ) of Lipofectamine and analyzed by FRET flow cytometry after a 24-h treatment. To perform quantification of uptake, protein cell lysates were labeled with AF647 and membranes of EVs with CV Claret. Arbitrary gates representing different levels of uptake (low, L ; medium, M ; high, H ; unstained cells, U ) are shown. The percentage of cells in each of the gates shown is depicted in the corresponding individual table below each flow cytometry plot ( n = 3, average ± S.D., 40,000 cells/experiment were analyzed). A , FRET tau biosensor cells do not show FRET when treated with Lipofectamine vehicle only (right gates, shaded green ) and show no far-red signal because no exosome-like EVs or cell lysates were a dded. B–E , none of the treatments with labeled WT exosome-like EVs or WT cell lysates induces FRET. However, far-red analysis of the uptake shows that the tau biosensor cells internalized proteins in cell lysates and whole EVs from WT samples in the absence of Lipofectamine ( D and E ), but uptake increased with Lipofectamine ( B and C ). F and G , Tau biosensor cells treated with Tg cell lysates or EVs in the presence of Lipofectamine. A strong FRET signal is detected with AF647 Tg cell lysates ( F ) and CV Claret Tg exosome-like EVs ( G ). In the presence of Lipofectamine, the majority of cells reached a high level of uptake. Consequently, the majority of cells showing FRET are those with the highest level of uptake of far-red labeled Tg sample. H and I , no FRET events are detected with AF647 Tg cell lysates ( H ) or CV Claret Tg EVs ( I ) in the absence of Lipofectamine. The majority of cells only reached a medium level of uptake. J and K , treatments with P301L Tg cell lysates and exosome-like EVs without the far-red label were used as a positive control.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Levels of uptake of exosome-like EVs and proteins in cell lysates determine the generation of the FRET signal. Tau biosensor cells were treated in the presence ( B , C , F , G , J , and K ) or absence ( D , E , H , and I ) of Lipofectamine and analyzed by FRET flow cytometry after a 24-h treatment. To perform quantification of uptake, protein cell lysates were labeled with AF647 and membranes of EVs with CV Claret. Arbitrary gates representing different levels of uptake (low, L ; medium, M ; high, H ; unstained cells, U ) are shown. The percentage of cells in each of the gates shown is depicted in the corresponding individual table below each flow cytometry plot ( n = 3, average ± S.D., 40,000 cells/experiment were analyzed). A , FRET tau biosensor cells do not show FRET when treated with Lipofectamine vehicle only (right gates, shaded green ) and show no far-red signal because no exosome-like EVs or cell lysates were a dded. B–E , none of the treatments with labeled WT exosome-like EVs or WT cell lysates induces FRET. However, far-red analysis of the uptake shows that the tau biosensor cells internalized proteins in cell lysates and whole EVs from WT samples in the absence of Lipofectamine ( D and E ), but uptake increased with Lipofectamine ( B and C ). F and G , Tau biosensor cells treated with Tg cell lysates or EVs in the presence of Lipofectamine. A strong FRET signal is detected with AF647 Tg cell lysates ( F ) and CV Claret Tg exosome-like EVs ( G ). In the presence of Lipofectamine, the majority of cells reached a high level of uptake. Consequently, the majority of cells showing FRET are those with the highest level of uptake of far-red labeled Tg sample. H and I , no FRET events are detected with AF647 Tg cell lysates ( H ) or CV Claret Tg EVs ( I ) in the absence of Lipofectamine. The majority of cells only reached a medium level of uptake. J and K , treatments with P301L Tg cell lysates and exosome-like EVs without the far-red label were used as a positive control.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Flow Cytometry, Cytometry, Labeling, Positive Control

    Far-red fluorescent labeling of proteins and exosome-like EVs. A , the strategy for labeling of free proteins and protein aggregates present in brain cell lysates. Amine-reactive Alexa Fluor 647 (AF647) labels the available N termini. Unreacted label is discarded by size exclusion chromatography, which recovers only AF647-labeled proteins and aggregates above 40 kDa. RT , room temperature. B , outline of the labeling of EV membranes with a lipophilic far-red dye (Cell Vue Claret). Lipophilic Cell Vue Claret incorporates with high affinity into the lipid bilayer of EV membranes. Traces of unbound dye are chelated with 1% BSA, and labeled exosome-like EVs are further purified by ultracentrifugation and PBS washes. It is important to note that the fluorescent label is in the membranes of exosome-like EVs, not the proteins.

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Far-red fluorescent labeling of proteins and exosome-like EVs. A , the strategy for labeling of free proteins and protein aggregates present in brain cell lysates. Amine-reactive Alexa Fluor 647 (AF647) labels the available N termini. Unreacted label is discarded by size exclusion chromatography, which recovers only AF647-labeled proteins and aggregates above 40 kDa. RT , room temperature. B , outline of the labeling of EV membranes with a lipophilic far-red dye (Cell Vue Claret). Lipophilic Cell Vue Claret incorporates with high affinity into the lipid bilayer of EV membranes. Traces of unbound dye are chelated with 1% BSA, and labeled exosome-like EVs are further purified by ultracentrifugation and PBS washes. It is important to note that the fluorescent label is in the membranes of exosome-like EVs, not the proteins.

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Labeling, Size-exclusion Chromatography, Purification

    Phosphorylation-dependent tau epitopes are present at very low levels in exosome-like EVs isolated from mouse brains. Shown are Western blotting analysis of sucrose fractions F1-F6, testing different AD-associated phospho-tau epitopes. 20 μg of total protein was loaded for F3 exosome-like EVs and whole cell lysate controls. Error bars represent S.E. ( n = 3). *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

    doi: 10.1074/jbc.M115.709485

    Figure Lengend Snippet: Phosphorylation-dependent tau epitopes are present at very low levels in exosome-like EVs isolated from mouse brains. Shown are Western blotting analysis of sucrose fractions F1-F6, testing different AD-associated phospho-tau epitopes. 20 μg of total protein was loaded for F3 exosome-like EVs and whole cell lysate controls. Error bars represent S.E. ( n = 3). *, p

    Article Snippet: After lysate treatments, the media of WT and Tg cell cultures were changed with exosome collection medium composed of DMEM (Life Technologies) supplemented with 5% exosome-depleted fetal bovine serum, 100 units/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mm GlutaMAX (Life Technologies).

    Techniques: Isolation, Western Blot

    Transient expression of OCT4 produces a change in methylation state of the endogenous OCT4 and NANOG promoters in the transfected human skin keratinocytes (HSK-OCT4-GFP). Shown are methylation states of CpG islands −929 to +421 around the transcription start site of the endogenous OCT4 promoter and upstream areas −1091 to −299 of the NANOG promoter for the NTERA-2 cell line (positive control), human skin keratinocytes (HSK-GFP) 48 hours after transient transfection with pcDNA3-GFP (negative control), and human skin keratinocytes (HSK-OCT4-GFP) 48 hours after transient transfection with pcDNA3-OCT4-GFP. Transfected keratinocytes were sorted from untransfected keratinocytes based on GFP; thus the analysis was done only on the transfected cells. Note, demethylation of OCT4 was consistent at +74 and +80, and demethylation of NANOG was consistent at −649, −611, −565, −516, and −378. Numbers relative to translational start site.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Transient expression of OCT4 produces a change in methylation state of the endogenous OCT4 and NANOG promoters in the transfected human skin keratinocytes (HSK-OCT4-GFP). Shown are methylation states of CpG islands −929 to +421 around the transcription start site of the endogenous OCT4 promoter and upstream areas −1091 to −299 of the NANOG promoter for the NTERA-2 cell line (positive control), human skin keratinocytes (HSK-GFP) 48 hours after transient transfection with pcDNA3-GFP (negative control), and human skin keratinocytes (HSK-OCT4-GFP) 48 hours after transient transfection with pcDNA3-OCT4-GFP. Transfected keratinocytes were sorted from untransfected keratinocytes based on GFP; thus the analysis was done only on the transfected cells. Note, demethylation of OCT4 was consistent at +74 and +80, and demethylation of NANOG was consistent at −649, −611, −565, −516, and −378. Numbers relative to translational start site.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Expressing, Methylation, Transfection, Positive Control, Negative Control

    Transient expression of OCT4 induces endogenous expression of OCT4 target genes in human skin keratinocytes. Shown in top four gels are RT-PCR gels for expression of OCT4 and the endogenous OCT4 target genes, SOX2, NANOG, and REX-1, from 1 through 5 days after transient transfection with pcDNA3-OCT4. Bottom gel demonstrates that untransfected HSKs do not express OCT4 RNA. 100bp = 100 base pair ladder.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Transient expression of OCT4 induces endogenous expression of OCT4 target genes in human skin keratinocytes. Shown in top four gels are RT-PCR gels for expression of OCT4 and the endogenous OCT4 target genes, SOX2, NANOG, and REX-1, from 1 through 5 days after transient transfection with pcDNA3-OCT4. Bottom gel demonstrates that untransfected HSKs do not express OCT4 RNA. 100bp = 100 base pair ladder.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection

    Human skin keratinocytes (HSKs) transiently transfected with pcDNA3-OCT4 can be differentiation into neuronal-like cell types. A) Phase image of HSKs transfected with pcDNA3-OCT4 for 48 hrs, then grown for 12 days in KSFM. B) Phase image of HSKs transfected with pc-DNA3-OCT4 for 48 hrs, then grown for 12 days in neuronal medium from protocol #1. C) RT-PCR showing expression of neuronal nuclear Fox-3 splicing protein (NeuN), neuron-specific class III β Tubulin (Tuj1), tyrosine hydroxylase (TH), keratin 14 (K14), and GAPDH. Lanes 1 = Untransfected HSKs grown for 22 days in KSFM; 2 = Untransfected HSKs grown for 22 days in neuronal medium from protocol #1; 3 = Neuroepithelioma SK-N-MC cell line; 4 = HSKs transfected with OCT4 for 48 hrs, then grown for 22 days in neuronal medium #1; 5 = HSKs transfected with OCT4 for 48 hrs, then grown for 12 days in neuronal medium #2. D) Western blots showing expression of NeuN protein. Lanes 1 = HSKs transfected with OCT4 for 48 hrs, then grown for 22 days in neuronal medium #1; 2 = HSKs transfected with OCT4 for 48 hrs and grown in KSFM for 22 days; 3 = untransfected HSKs grown in neuronal medium #1 for 22 days; 4 = untransfected HSKs grown in KSFM for 22 days. Note, only cells in lane 1 show expression of NeuN protein. KSFM = keratinocyte serum free medium.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Human skin keratinocytes (HSKs) transiently transfected with pcDNA3-OCT4 can be differentiation into neuronal-like cell types. A) Phase image of HSKs transfected with pcDNA3-OCT4 for 48 hrs, then grown for 12 days in KSFM. B) Phase image of HSKs transfected with pc-DNA3-OCT4 for 48 hrs, then grown for 12 days in neuronal medium from protocol #1. C) RT-PCR showing expression of neuronal nuclear Fox-3 splicing protein (NeuN), neuron-specific class III β Tubulin (Tuj1), tyrosine hydroxylase (TH), keratin 14 (K14), and GAPDH. Lanes 1 = Untransfected HSKs grown for 22 days in KSFM; 2 = Untransfected HSKs grown for 22 days in neuronal medium from protocol #1; 3 = Neuroepithelioma SK-N-MC cell line; 4 = HSKs transfected with OCT4 for 48 hrs, then grown for 22 days in neuronal medium #1; 5 = HSKs transfected with OCT4 for 48 hrs, then grown for 12 days in neuronal medium #2. D) Western blots showing expression of NeuN protein. Lanes 1 = HSKs transfected with OCT4 for 48 hrs, then grown for 22 days in neuronal medium #1; 2 = HSKs transfected with OCT4 for 48 hrs and grown in KSFM for 22 days; 3 = untransfected HSKs grown in neuronal medium #1 for 22 days; 4 = untransfected HSKs grown in KSFM for 22 days. Note, only cells in lane 1 show expression of NeuN protein. KSFM = keratinocyte serum free medium.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

    Transient expression of OCT4 produces an overall decrease in global methylation in the transfected human skin keratinocytes (HSK). Shown is a graph depicting percent global methylation in four cellular groups: Untransfected HSKs (HSK); HSKs transiently transfected with pcDNA3-GFP (HSK-GFP) for 48 hours; HSKs transiently transfected with pcDNA3-OCT4-GFP fusion (HSK-OCT4) for 48 hours; and the NTERA-2 positive control cell line that continually expresses OCT4. Note, transfection of OCT4 significantly decreased the global methylation state of HSK (p=0.002) as compared to transfection with GFP alone.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Transient expression of OCT4 produces an overall decrease in global methylation in the transfected human skin keratinocytes (HSK). Shown is a graph depicting percent global methylation in four cellular groups: Untransfected HSKs (HSK); HSKs transiently transfected with pcDNA3-GFP (HSK-GFP) for 48 hours; HSKs transiently transfected with pcDNA3-OCT4-GFP fusion (HSK-OCT4) for 48 hours; and the NTERA-2 positive control cell line that continually expresses OCT4. Note, transfection of OCT4 significantly decreased the global methylation state of HSK (p=0.002) as compared to transfection with GFP alone.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Expressing, Methylation, Transfection, Positive Control

    Human skin keratinocytes (HSKs) transiently transfected with pcDNA3-OCT4 can be differentiated into myofibroblasts or smooth muscle-type cells. A) Phase image of HSKs grown in KSFM for 12 days. B) Phase image of OCT4-transfected HSKs grown in smooth muscle differentiation medium for 11 days. C) RT-PCR showing expression of calponin, SM22α, and keratin 14 (K14). Lanes 1 = HSKs transfected with OCT4 for 48 hrs, then grown in mesenchymal differentiation medium for 12 days; 2 = HSKs transfected with OCT4 for 48 hrs and grown in KSFM for 12 days; 3 = untransfected HSKs grown in KSFM for 12 days; 4 = human aorta cDNA positive control. E) Western blot analysis of vimentin, SM22α, myocardin, and actin proteins. Lanes 1 = HSKs transfected with OCT4 for 48 hrs, then grown in mesenchymal differentiation medium for 12 days; 2 = HSKs transfected with OCT4 for 48 hrs and grown in KSFM for 12 days; 3 = untransfected HSKs grown in KSFM for 12 days; 4 = WPMY-1 myofibroblast cell line. Note, myocardin has three isoforms, 73 kD, 95.7 kD, and 101 kD 34 . KSFM = keratinocyte serum free medium.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Human skin keratinocytes (HSKs) transiently transfected with pcDNA3-OCT4 can be differentiated into myofibroblasts or smooth muscle-type cells. A) Phase image of HSKs grown in KSFM for 12 days. B) Phase image of OCT4-transfected HSKs grown in smooth muscle differentiation medium for 11 days. C) RT-PCR showing expression of calponin, SM22α, and keratin 14 (K14). Lanes 1 = HSKs transfected with OCT4 for 48 hrs, then grown in mesenchymal differentiation medium for 12 days; 2 = HSKs transfected with OCT4 for 48 hrs and grown in KSFM for 12 days; 3 = untransfected HSKs grown in KSFM for 12 days; 4 = human aorta cDNA positive control. E) Western blot analysis of vimentin, SM22α, myocardin, and actin proteins. Lanes 1 = HSKs transfected with OCT4 for 48 hrs, then grown in mesenchymal differentiation medium for 12 days; 2 = HSKs transfected with OCT4 for 48 hrs and grown in KSFM for 12 days; 3 = untransfected HSKs grown in KSFM for 12 days; 4 = WPMY-1 myofibroblast cell line. Note, myocardin has three isoforms, 73 kD, 95.7 kD, and 101 kD 34 . KSFM = keratinocyte serum free medium.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Western Blot

    Transient expression of OCT4 in human keratinocytes. A) Immunofluorescent image showing that untransfected human skin keratinocytes do not express OCT4 (20x). Cells were stained with both DAPI (blue) to identify all nuclei and with antibody to OCT4 (red). B) Immunofluorescent image of keratinocytes 48 hours after transient transfection with pcDNA-OCT4 (40x). Nuclei are identified by DAPI (blue). Transfected cells are identified by antibody to OCT4 (red/pink). C) Graph depicting temporal expression of OCT4 in human keratinocytes. Shown is the percentage of cells expressing OCT4 protein, from 1 through 6 days after transient transfection with pcDNA3-OCT4.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Transient expression of OCT4 in human keratinocytes. A) Immunofluorescent image showing that untransfected human skin keratinocytes do not express OCT4 (20x). Cells were stained with both DAPI (blue) to identify all nuclei and with antibody to OCT4 (red). B) Immunofluorescent image of keratinocytes 48 hours after transient transfection with pcDNA-OCT4 (40x). Nuclei are identified by DAPI (blue). Transfected cells are identified by antibody to OCT4 (red/pink). C) Graph depicting temporal expression of OCT4 in human keratinocytes. Shown is the percentage of cells expressing OCT4 protein, from 1 through 6 days after transient transfection with pcDNA3-OCT4.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Expressing, Staining, Transfection

    Transient expression of OCT4 allows human keratinocytes (HSKs) to be directed to differentiate into contractile cells. Cells were embedded in collagen type I in 12-well culture plates and allowed to contract the collagen gels for four days. A) Photographs of collagen gels at 3 days after cells were embedded into the gels. B) Graph showing amount of contraction by the various cells over 4 days. Note, HSKs transfected with OCT4 for 48 hrs, then grown in mesenchymal differentiation medium for 12 days contracted the collagen gels as well as the fibroblasts and the WPMY-1 myofibroblast cell line contracted the gels, whereas untransfected HSKs showed no contractility whether grown in KSFM or in mesenchymal differentiation medium. 1 = HSKs transfected with OCT4 for 48 hrs, grown in mesenchymal differentiation medium for 12 days, then embedded in the collagen type I gel; 2 = untransfected HSKs grown in mesenchymal differentiation medium for 12 days, then embedded in the collagen gel; 3 = untransfected HSKs grown in KSFM for 12 days, then embedded in the collagen gel; 4 = primary human fibroblasts grown in DMEM then embedded in the collagen gel; 5 = WPMY-1 myofibroblast cell line grown in DMEM then embedded in the collagen gel. KSFM = keratinocyte serum free medium.

    Journal: Gene therapy

    Article Title: Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway

    doi: 10.1038/gt.2010.148

    Figure Lengend Snippet: Transient expression of OCT4 allows human keratinocytes (HSKs) to be directed to differentiate into contractile cells. Cells were embedded in collagen type I in 12-well culture plates and allowed to contract the collagen gels for four days. A) Photographs of collagen gels at 3 days after cells were embedded into the gels. B) Graph showing amount of contraction by the various cells over 4 days. Note, HSKs transfected with OCT4 for 48 hrs, then grown in mesenchymal differentiation medium for 12 days contracted the collagen gels as well as the fibroblasts and the WPMY-1 myofibroblast cell line contracted the gels, whereas untransfected HSKs showed no contractility whether grown in KSFM or in mesenchymal differentiation medium. 1 = HSKs transfected with OCT4 for 48 hrs, grown in mesenchymal differentiation medium for 12 days, then embedded in the collagen type I gel; 2 = untransfected HSKs grown in mesenchymal differentiation medium for 12 days, then embedded in the collagen gel; 3 = untransfected HSKs grown in KSFM for 12 days, then embedded in the collagen gel; 4 = primary human fibroblasts grown in DMEM then embedded in the collagen gel; 5 = WPMY-1 myofibroblast cell line grown in DMEM then embedded in the collagen gel. KSFM = keratinocyte serum free medium.

    Article Snippet: Cells were plated at a concentration of 8×104 cells/ml in keratinocyte serum free medium (KSFM, Invitrogen) + 1.5% Antibiotic-antimycotic on culture dishes coated with 0.25μg/cm2 collagen type IV (Collaborative Biomedical).

    Techniques: Expressing, Transfection

    The effects of collagen I gel concentrations on CD133 + cell migration in the 3D μ-slide assay. (A) After 24 hour culture of human UCB CD133 + cells in StemSpan medium and SCF, Flt-3 ligand, IL-6 and TPO, cells were suspended in 0.5 mg/ml, 1 mg/ml or 3 mg/ml collagen I gel solutions and seeded into the central chamber of the 3D chemotaxis μ-slides. A chemokine gradient was established using 1 μg/ml CXCL12. The cells were imaged over 22 h by timelapse microscopy migration and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug in for (a) accumulated distance, (b) velocity, (c) displacement of the center of mass and (d) forward migration index. Values are means ± SEM (n ≥ 3independent experiments). Variability amongst independent experiments was increased when 3 mg/ml collagen I gels were compared with 0.5 or 1 mg/ml collagen I gels. This is exemplified when comparing 1 mg/ml and 3 mg/ml collagen I gels respectively for the ranges in the accumulated distance (1012 μm to 1451 μm vs. 350 μm to 1467 μm respectively), velocity (0.77 to 1.16 vs. 0.27 to 1.18 μm/min respectively), displacement of center of mass (111.80 to 115.43 vs. 26.94 to 256.20 μm respectively) and forward migration index (0.100 to 0.110 vs. 0.060 to 0.220 respectively). (B) Microscopic images (× 4 magnification) of CD133 + cells in (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 3 mg/ml collagen 1 gels after seeding into the central chamber of the 3D chemotaxis μ-slide and demonstrating the lack of uniformity of collagen I fibers with 3 mg/ml collagen I gels.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: The effects of collagen I gel concentrations on CD133 + cell migration in the 3D μ-slide assay. (A) After 24 hour culture of human UCB CD133 + cells in StemSpan medium and SCF, Flt-3 ligand, IL-6 and TPO, cells were suspended in 0.5 mg/ml, 1 mg/ml or 3 mg/ml collagen I gel solutions and seeded into the central chamber of the 3D chemotaxis μ-slides. A chemokine gradient was established using 1 μg/ml CXCL12. The cells were imaged over 22 h by timelapse microscopy migration and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug in for (a) accumulated distance, (b) velocity, (c) displacement of the center of mass and (d) forward migration index. Values are means ± SEM (n ≥ 3independent experiments). Variability amongst independent experiments was increased when 3 mg/ml collagen I gels were compared with 0.5 or 1 mg/ml collagen I gels. This is exemplified when comparing 1 mg/ml and 3 mg/ml collagen I gels respectively for the ranges in the accumulated distance (1012 μm to 1451 μm vs. 350 μm to 1467 μm respectively), velocity (0.77 to 1.16 vs. 0.27 to 1.18 μm/min respectively), displacement of center of mass (111.80 to 115.43 vs. 26.94 to 256.20 μm respectively) and forward migration index (0.100 to 0.110 vs. 0.060 to 0.220 respectively). (B) Microscopic images (× 4 magnification) of CD133 + cells in (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 3 mg/ml collagen 1 gels after seeding into the central chamber of the 3D chemotaxis μ-slide and demonstrating the lack of uniformity of collagen I fibers with 3 mg/ml collagen I gels.

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Migration, Chemotaxis Assay, Microscopy, Software

    Expression of CXCR4 on human cord blood CD133 + cells. (A (a)) Representative FACS dot plots of purified human UCB CD133 + cells (averaging 94 + 2% purity for the optimization and validation experiments) showing dual staining for respective isotype controls mIgG2a-APC vs. mIgG2b-PE or CD133-PE and CD34-APC antibodies after 24 hour culture in StemSpan serum free medium with SCF, Flt-3 ligand, IL-6 and TPO and hence at the commencement of the migration assays. (A (b to e)) Representative FACS histograms of cells from (A (a)) after mIgG2b-PE and CD133-PE or mIgG2a-APC and CXCR4-APC staining. (B) Median fluorescence intensities (MFIs) of n = 7 CD133 + cell preparations stained with the isotype control or the CXCR4 antibody with individual experiments represented by scatter plots and the median value being represented by the horizontal bar. For CXCR4 staining, MFIs averaged (mean ± SEM) 726.4 ± 153.3 compared to 38.0 + 8.5 for the negative isotype control.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: Expression of CXCR4 on human cord blood CD133 + cells. (A (a)) Representative FACS dot plots of purified human UCB CD133 + cells (averaging 94 + 2% purity for the optimization and validation experiments) showing dual staining for respective isotype controls mIgG2a-APC vs. mIgG2b-PE or CD133-PE and CD34-APC antibodies after 24 hour culture in StemSpan serum free medium with SCF, Flt-3 ligand, IL-6 and TPO and hence at the commencement of the migration assays. (A (b to e)) Representative FACS histograms of cells from (A (a)) after mIgG2b-PE and CD133-PE or mIgG2a-APC and CXCR4-APC staining. (B) Median fluorescence intensities (MFIs) of n = 7 CD133 + cell preparations stained with the isotype control or the CXCR4 antibody with individual experiments represented by scatter plots and the median value being represented by the horizontal bar. For CXCR4 staining, MFIs averaged (mean ± SEM) 726.4 ± 153.3 compared to 38.0 + 8.5 for the negative isotype control.

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Expressing, FACS, Purification, Staining, Migration, Fluorescence

    Comparison of chemotactic and chemokinetic responses of pooled versus non-pooled human cord blood CD133 + cells. Human CD133 + cells were isolated from umbilical cord blood and cultured as individual units (non-pooled; n = 7) or as pooled units (pooled; n = 8) in StemSpan medium containing SCF, Flt-3 ligand, IL-6 and TPO for 24 h before encapsulation into 1 mg/ml collagen I gel and seeding into the central chamber of a 3D chemotaxis μ-slide. Cell migration was tracked using timelapse microscopy and Image J software over 22 h in the presence of 1 μg/ml CXCL12. The chemotaxis and migration tool plug in was used to quantify (a) the percentage of cells migrating, (b) the displacement of the center of mass, (c) forward migration index, (d) directionality, (e) velocity, and (f) accumulated distance. Scatter plots represent individual experiments and the median for each group is represented as a horizontal bar. While individual or pooled cell samples varied in their migratory capacities between experiments, the median values were not significantly different. This is also exemplified by mean + SEM values for forward migration index (pooled 0.155 ± 0.030, range 0.04–0.30 and non-pooled 0.16 ± 0.02, range 0.10–0.27; p = 0.9 n = 8 and 7 respectively) and cell velocity (pooled 1.07 ± 0.04, range 0.92–1.29 μm/min and non-pooled 1.06 ± 0.11, range 0.77–0.11 μm/min; p = 0.5) (Mann Whitney test).

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: Comparison of chemotactic and chemokinetic responses of pooled versus non-pooled human cord blood CD133 + cells. Human CD133 + cells were isolated from umbilical cord blood and cultured as individual units (non-pooled; n = 7) or as pooled units (pooled; n = 8) in StemSpan medium containing SCF, Flt-3 ligand, IL-6 and TPO for 24 h before encapsulation into 1 mg/ml collagen I gel and seeding into the central chamber of a 3D chemotaxis μ-slide. Cell migration was tracked using timelapse microscopy and Image J software over 22 h in the presence of 1 μg/ml CXCL12. The chemotaxis and migration tool plug in was used to quantify (a) the percentage of cells migrating, (b) the displacement of the center of mass, (c) forward migration index, (d) directionality, (e) velocity, and (f) accumulated distance. Scatter plots represent individual experiments and the median for each group is represented as a horizontal bar. While individual or pooled cell samples varied in their migratory capacities between experiments, the median values were not significantly different. This is also exemplified by mean + SEM values for forward migration index (pooled 0.155 ± 0.030, range 0.04–0.30 and non-pooled 0.16 ± 0.02, range 0.10–0.27; p = 0.9 n = 8 and 7 respectively) and cell velocity (pooled 1.07 ± 0.04, range 0.92–1.29 μm/min and non-pooled 1.06 ± 0.11, range 0.77–0.11 μm/min; p = 0.5) (Mann Whitney test).

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Isolation, Cell Culture, Chemotaxis Assay, Migration, Microscopy, Software, MANN-WHITNEY

    CD133 + cell chemotaxis and chemokinesis as a function of time. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient as illustrated in Fig. 1 . Cell migration was imaged for 22 h by timelapse microscopy and tracked and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in after 4, 10, 18 and 22 h. (A) shows representative trajectory plots (a) 4 h, (b) 10 h, (c) 18 h and (d) 22 h (the blue cross shows the displacement of the center of mass) of cells exposed to 1 μg/ml CXCL12. The red and black lines indicate whether the cells finished their migration path below (towards 1 μg/ml CXCL12) or above their starting point on the x axis. The Rayleigh test indicate that the distribution of the cell end points was significantly inhomogeneous at all time points (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (p = 4.0 × 10 − 3 , p = 2.9 × 10 − 5 , p = 6.7 × 10 − 7 and 2.0 × 10 − 8 for (a)–(d) respectively. (B) shows the quantitative data for (a,b) accumulated distance, (c,d) velocity, (e,f) displacement of the center of mass, and (g,h) forward migration index, of cells exposed to 0 μg/ml CXCL12 (a,c,e,g) or 1 μg/ml CXCL12 (b,d,f,h) respectively. Values are means ± SEM for n = 3 independent experiments.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: CD133 + cell chemotaxis and chemokinesis as a function of time. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient as illustrated in Fig. 1 . Cell migration was imaged for 22 h by timelapse microscopy and tracked and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in after 4, 10, 18 and 22 h. (A) shows representative trajectory plots (a) 4 h, (b) 10 h, (c) 18 h and (d) 22 h (the blue cross shows the displacement of the center of mass) of cells exposed to 1 μg/ml CXCL12. The red and black lines indicate whether the cells finished their migration path below (towards 1 μg/ml CXCL12) or above their starting point on the x axis. The Rayleigh test indicate that the distribution of the cell end points was significantly inhomogeneous at all time points (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (p = 4.0 × 10 − 3 , p = 2.9 × 10 − 5 , p = 6.7 × 10 − 7 and 2.0 × 10 − 8 for (a)–(d) respectively. (B) shows the quantitative data for (a,b) accumulated distance, (c,d) velocity, (e,f) displacement of the center of mass, and (g,h) forward migration index, of cells exposed to 0 μg/ml CXCL12 (a,c,e,g) or 1 μg/ml CXCL12 (b,d,f,h) respectively. Values are means ± SEM for n = 3 independent experiments.

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Chemotaxis Assay, Cell Culture, Migration, Microscopy, Software

    The 3D μ-slide chemotaxis assay for human cord blood CD133 + hematopoietic stem/progenitor cells. (A) After 24 hour culture in StemSpan medium plus SCF, Flt-3 ligand, IL-6 and TPO, (a) human UCB CD133 + cells in a collagen I gel solution (1 mg/ml) were seeded into the central 3D μ-slide chamber prior to (b) the addition of StemSpan serum free medium alone (C0) or with a 1 μg/ml CXCL12 (C100) gradient plus or minus AMD3100 and cells captured by timelapse microscopy at 3–5 minute intervals. (B) Trajectory plots illustrate migrated cells at 22 h minus (a) or plus a CXCL12 gradient (b) or with CXCL12 in the presence of 10 μM AMD3100 (c). The red and black lines indicate whether the cells finished their migration path below or above their starting point on the x axis. Rayleigh test p values of p = 0.17, p = 5.7 × 10 − 8 and p = 0.054 for (a), (b) and (c) respectively indicate that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 alone (b). (C) Diagrammatic representation of a trajectory plot demonstrating methods for quantitating chemotactic and chemokinetic parameters. The diagrams in Figs. 1A and C were captured from the movie describing the assay system (MV_25_chemotaxis.flv) on the Ibidi website ( http://ibidi.com/support/movies/mv25/ ).

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: The 3D μ-slide chemotaxis assay for human cord blood CD133 + hematopoietic stem/progenitor cells. (A) After 24 hour culture in StemSpan medium plus SCF, Flt-3 ligand, IL-6 and TPO, (a) human UCB CD133 + cells in a collagen I gel solution (1 mg/ml) were seeded into the central 3D μ-slide chamber prior to (b) the addition of StemSpan serum free medium alone (C0) or with a 1 μg/ml CXCL12 (C100) gradient plus or minus AMD3100 and cells captured by timelapse microscopy at 3–5 minute intervals. (B) Trajectory plots illustrate migrated cells at 22 h minus (a) or plus a CXCL12 gradient (b) or with CXCL12 in the presence of 10 μM AMD3100 (c). The red and black lines indicate whether the cells finished their migration path below or above their starting point on the x axis. Rayleigh test p values of p = 0.17, p = 5.7 × 10 − 8 and p = 0.054 for (a), (b) and (c) respectively indicate that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 alone (b). (C) Diagrammatic representation of a trajectory plot demonstrating methods for quantitating chemotactic and chemokinetic parameters. The diagrams in Figs. 1A and C were captured from the movie describing the assay system (MV_25_chemotaxis.flv) on the Ibidi website ( http://ibidi.com/support/movies/mv25/ ).

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Chemotaxis Assay, Microscopy, Migration

    CXCL12 induces human cord blood CD133 + hematopoietic stem/progenitor cell chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient. (A) The results of 8 independent experiments are displayed as median values (horizontal bar) for individual experiments (scatter plots). Cell migration was tracked using Image J manual tracking software for 22 h and chemotactic and chemokinetic parameters quantified with the chemotaxis and migration tool plug-in for (a) the Rayleigh p value, (b) displacement of the center of mass (blue cross in Figs. 1 B and C), (c) forward migration index, (d) directionality, (e) velocity and (f) accumulated distance. Interestingly, there were significant differences in chemotaxis between the chambers containing medium alone and those containing CXCL12, with the following respective changes (mean ± SEM) in Rayleigh values (0.336 ± 0.073 vs. 0.001 ± 0.001; p = 0.0002), displacement of center of mass (10.82 ± 11.25 vs. 189.00 ± 44.26; p = 0.0002) and forward migration index (0.015 ± 0.008 vs. 0.126 ± 0.024; p = 0.0004) over 8 independent experiments, but no significant difference in cell velocity or accumulated distance without or with a CXCL12 gradient (p = 0.7128 and p = 0.2345 respectively). Statistics for chemotactic and chemokinetic data were calculated using the Mann–Whitney test with *p

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: CXCL12 induces human cord blood CD133 + hematopoietic stem/progenitor cell chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient. (A) The results of 8 independent experiments are displayed as median values (horizontal bar) for individual experiments (scatter plots). Cell migration was tracked using Image J manual tracking software for 22 h and chemotactic and chemokinetic parameters quantified with the chemotaxis and migration tool plug-in for (a) the Rayleigh p value, (b) displacement of the center of mass (blue cross in Figs. 1 B and C), (c) forward migration index, (d) directionality, (e) velocity and (f) accumulated distance. Interestingly, there were significant differences in chemotaxis between the chambers containing medium alone and those containing CXCL12, with the following respective changes (mean ± SEM) in Rayleigh values (0.336 ± 0.073 vs. 0.001 ± 0.001; p = 0.0002), displacement of center of mass (10.82 ± 11.25 vs. 189.00 ± 44.26; p = 0.0002) and forward migration index (0.015 ± 0.008 vs. 0.126 ± 0.024; p = 0.0004) over 8 independent experiments, but no significant difference in cell velocity or accumulated distance without or with a CXCL12 gradient (p = 0.7128 and p = 0.2345 respectively). Statistics for chemotactic and chemokinetic data were calculated using the Mann–Whitney test with *p

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Chemotaxis Assay, Cell Culture, Migration, Software, MANN-WHITNEY

    AMD3100 inhibits chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 1 μg/ml CXCL12 to form a chemokine gradient with or without AMD3100. Histograms Illustrate the effect of adding 0, 1 μM or 10 μM AMD3100 with 1 μg/ml CXCL12 for the chemotactic or chemokinetic responses of forward migration index (A) and displacement of the center of mass (B) or velocity (C) and accumulated distance (D). Controls contained media alone. Values are means ± SEM for n ≥ 3 independent experiments. Notably, with the addition of AMD3100, there was a significant decrease in the Rayleigh test p values (E), in displacement of the center of mass (mean ± SEM values of 126.5 μm ± 21.1 for no AMD3100 to 24.7 μm ± 18.1 for 1 μM AMD3100 and 7.5 μm ± 10.6 for 10 μM AMD3100) and in forward migration index (mean ± SEM values of 0.11 ± 0.02 with no AMD3100 and 0.01 ± 0.01 for 1 μM AMD3100 and 0.01 ± 0.01 for 10 μM AMD3100) towards CXCL12 compared to absent AMD3100 (p

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: AMD3100 inhibits chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 1 μg/ml CXCL12 to form a chemokine gradient with or without AMD3100. Histograms Illustrate the effect of adding 0, 1 μM or 10 μM AMD3100 with 1 μg/ml CXCL12 for the chemotactic or chemokinetic responses of forward migration index (A) and displacement of the center of mass (B) or velocity (C) and accumulated distance (D). Controls contained media alone. Values are means ± SEM for n ≥ 3 independent experiments. Notably, with the addition of AMD3100, there was a significant decrease in the Rayleigh test p values (E), in displacement of the center of mass (mean ± SEM values of 126.5 μm ± 21.1 for no AMD3100 to 24.7 μm ± 18.1 for 1 μM AMD3100 and 7.5 μm ± 10.6 for 10 μM AMD3100) and in forward migration index (mean ± SEM values of 0.11 ± 0.02 with no AMD3100 and 0.01 ± 0.01 for 1 μM AMD3100 and 0.01 ± 0.01 for 10 μM AMD3100) towards CXCL12 compared to absent AMD3100 (p

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Chemotaxis Assay, Cell Culture, Migration

    The CD133 + cell chemotactic response to increasing concentrations of CXCL12. Human UCB CD133 + cells cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h were encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides. 0 μg/ml, 0.2 μg/ml, 1 μg/ml or 2 μg/ml CXCL12 were added to create a chemokine gradient. The cells were imaged over 22 h by timelapse microscopy and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in. (A) Representative trajectory plots after CD133 + cells were exposed to differing concentrations of CXCL12. The red and black lines indicate whether the cells finished their migration path below or above (towards differing CXCL12 concentrations) their starting point on the x axis. Rayleigh test p values were p = 0.16, p = 6.1 × 10 − 5 , p = 4.0 × 10 − 10 and p = 1.0 × 10 − 7 respectively for figures (a)–(d) indicating that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (b)–(d) and that this was most significant with 1 μg/ml (c). (B) shows the quantitative data for (a) displacement of center of mass (257.8 μm ± 112.9 for 1 μg/ml compared to no CXCL12 of 48.8 μm ± 8.9), (b) forward migration index (0.18 ± 0.05 for 1 μg/ml compared to no CXCL12 of 0.02 ± 0.00), (c) cell velocity and (d) accumulated distance as a function of CXCL12 concentration. Values are means ± SEM for n = 3 independent experiments.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: The CD133 + cell chemotactic response to increasing concentrations of CXCL12. Human UCB CD133 + cells cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h were encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides. 0 μg/ml, 0.2 μg/ml, 1 μg/ml or 2 μg/ml CXCL12 were added to create a chemokine gradient. The cells were imaged over 22 h by timelapse microscopy and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in. (A) Representative trajectory plots after CD133 + cells were exposed to differing concentrations of CXCL12. The red and black lines indicate whether the cells finished their migration path below or above (towards differing CXCL12 concentrations) their starting point on the x axis. Rayleigh test p values were p = 0.16, p = 6.1 × 10 − 5 , p = 4.0 × 10 − 10 and p = 1.0 × 10 − 7 respectively for figures (a)–(d) indicating that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (b)–(d) and that this was most significant with 1 μg/ml (c). (B) shows the quantitative data for (a) displacement of center of mass (257.8 μm ± 112.9 for 1 μg/ml compared to no CXCL12 of 48.8 μm ± 8.9), (b) forward migration index (0.18 ± 0.05 for 1 μg/ml compared to no CXCL12 of 0.02 ± 0.00), (c) cell velocity and (d) accumulated distance as a function of CXCL12 concentration. Values are means ± SEM for n = 3 independent experiments.

    Article Snippet: In nonexpansion experiments, pooled or individual freshly harvested or cryopreserved CD133+ cells were cultured at 5 × 105 cells/ml in StemSpan serum-free medium (Stem Cell Technologies, Grenoble, France) containing 100 ng/ml interleukin-6 (IL-6), 20 ng/ml thrombopoietin (TPO), 100 ng/ml stem cell factor (SCF), and 100 ng/ml FLT3 ligand (R & D Systems, Minneapolis, MN, USA) in 6–12 well tissue culture plates at 37 °C for 24 h prior to use ( ).

    Techniques: Cell Culture, Chemotaxis Assay, Microscopy, Software, Migration, Concentration Assay

    The 3 - D fluorescence spectra of BSA and HSA. A. The 3-D fluorescence spectra and corresponding contour diagrams of BSA (A) and BSA-HU (B). The concentration of protein was 5 μM and that of HU was 20 μM. B. The 3-D fluorescence spectra and corresponding contour diagrams of HSA (A) and HSA-HU (B) . The concentration of protein was 5 μM and that of HU was 20 μM.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: The 3 - D fluorescence spectra of BSA and HSA. A. The 3-D fluorescence spectra and corresponding contour diagrams of BSA (A) and BSA-HU (B). The concentration of protein was 5 μM and that of HU was 20 μM. B. The 3-D fluorescence spectra and corresponding contour diagrams of HSA (A) and HSA-HU (B) . The concentration of protein was 5 μM and that of HU was 20 μM.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Fluorescence, Concentration Assay

    FT- IR spectra of (A) BSA- HU system and (B) HSA- HU system.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: FT- IR spectra of (A) BSA- HU system and (B) HSA- HU system.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques:

    Synchronous fluorescence spectra of BSA and HSA. A. Synchronous fluorescence spectra of BSA-HU: For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 μM. The concentration of BSA was 5.0 μM. B. Synchronous fluorescence spectra of HSA-HU: (A) For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20, (f) 25 and (g) 30 μM. The concentration of HSA was 5.0 μM.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Synchronous fluorescence spectra of BSA and HSA. A. Synchronous fluorescence spectra of BSA-HU: For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20 and (f) 25 μM. The concentration of BSA was 5.0 μM. B. Synchronous fluorescence spectra of HSA-HU: (A) For Δλ = 60 nm. Concentration of HU: (a) 0, (b) 5, (c) 10, (d) 15, (e) 20, (f) 25 and (g) 30 μM. The concentration of HSA was 5.0 μM.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Fluorescence, Concentration Assay

    Absorbance spectra of BSA and HSA. A. Absorbance spectra of BSA, HU and HU-BSA system. BSA concentration was 5 μM (a). HU concentration for HU–BSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU. B. Absorbance spectra of HSA, HU and HU-HSA system. HSA concentration was 5 μM (a). HU concentration for HU–HSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Absorbance spectra of BSA and HSA. A. Absorbance spectra of BSA, HU and HU-BSA system. BSA concentration was 5 μM (a). HU concentration for HU–BSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU. B. Absorbance spectra of HSA, HU and HU-HSA system. HSA concentration was 5 μM (a). HU concentration for HU–HSA system was at 5 μM (b) and 10 μM (c). (x) is the concentration of 5 μM HU.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Concentration Assay

    Structure of BSA and HSA.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Structure of BSA and HSA.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques:

    Van’t Hoff plot with BSA and HSA. A. van’t Hoff plot for the binding of HU with BSA. B. van’t Hoff plot for the binding of HU with HSA.

    Journal: SpringerPlus

    Article Title: Elucidation of binding mechanism of hydroxyurea on serum albumins by different spectroscopic studies

    doi: 10.1186/2193-1801-3-360

    Figure Lengend Snippet: Van’t Hoff plot with BSA and HSA. A. van’t Hoff plot for the binding of HU with BSA. B. van’t Hoff plot for the binding of HU with HSA.

    Article Snippet: Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma Chemical Company, St. Louis, USA and used without purification.

    Techniques: Binding Assay

    Effects of cycloheximide on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 10 μM cycloheximide for 24 hours in DMEM containing 10% FBS without (Chx) or with 33% vitreous humor (V/Chx). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: Effects of cycloheximide on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 10 μM cycloheximide for 24 hours in DMEM containing 10% FBS without (Chx) or with 33% vitreous humor (V/Chx). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Expressing, SDS Page, Purification, Western Blot, Imaging

    Western blot analysis of ferritin H- and L-chains in the lysates of control (C) and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with (V) or without (C) 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 11; statistically different from corresponding C, * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: Western blot analysis of ferritin H- and L-chains in the lysates of control (C) and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with (V) or without (C) 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 11; statistically different from corresponding C, * P

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Western Blot, Cell Culture, SDS Page, Purification, Imaging

    ( A ) Phase contrast image of control (C) and vitreous-treated LEC (V). LEC were treated for 96 hours with DMEM containing 1% FBS and 33% of vitreous humor. Control LEC were grown in DMEM containing 1% FBS without vitreous humor. ( B ) Western blot analysis of aquaporin 0 expression in treated (V) and control LEC (C). Lens lysates containing 50 μg proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-aquaporin 0 polyclonal antibodies. Canine lens was dissected and the section containing lens fibers was sonicated. Fifty micrograms of protein sample of sonicated lens fiber cells was used as a positive control (St). Blot was evaluated with ChemiDoc MP Imaging System.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: ( A ) Phase contrast image of control (C) and vitreous-treated LEC (V). LEC were treated for 96 hours with DMEM containing 1% FBS and 33% of vitreous humor. Control LEC were grown in DMEM containing 1% FBS without vitreous humor. ( B ) Western blot analysis of aquaporin 0 expression in treated (V) and control LEC (C). Lens lysates containing 50 μg proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with rabbit anti-aquaporin 0 polyclonal antibodies. Canine lens was dissected and the section containing lens fibers was sonicated. Fifty micrograms of protein sample of sonicated lens fiber cells was used as a positive control (St). Blot was evaluated with ChemiDoc MP Imaging System.

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Western Blot, Expressing, SDS Page, Sonication, Positive Control, Imaging

    Western blot analysis of TfR1 in control (C)- and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with or without 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 9; statistically different from corresponding C, * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: Western blot analysis of TfR1 in control (C)- and vitreous-treated LEC (V). Confluent LEC were cultured in DMEM with 10% FBS with or without 33% (vol/vol) of vitreous humor for 24 or 96 hours. LEC lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blot was reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 9; statistically different from corresponding C, * P

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Western Blot, Cell Culture, SDS Page, Purification, Imaging

    Effects of actinomycin D on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 1 μg/1 mL actinomycin D for 24 hours in DMEM containing 10% FBS without (ActD) or with 33% vitreous humor (V/ActD). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: Effects of actinomycin D on expression of ferritin chains ( A ) and TfR1 ( B ) in LEC treated with vitreous humor (V) and control LEC (C). LEC were treated with 1 μg/1 mL actinomycin D for 24 hours in DMEM containing 10% FBS without (ActD) or with 33% vitreous humor (V/ActD). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. Purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by using Tukey's HSD test. Data represent the mean ± SEM; n = 3; statistically different from corresponding C, * P

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Expressing, SDS Page, Purification, Western Blot, Imaging

    Western blot analysis of ferritin H- and L-chains in the lysates of LEC treated with differentially processed fractions of vitreous humor. LEC were treated for 24 hours without (C) or with 33% of vitreous humor (V) and with equivalent amount of differentially processed vitreous fractions, in DMEM containing 10% FBS. Vf, vitreous humor filtered through glass wool; Va, acetone precipitate of Vf dissolved in complete DMEM; Vmf, Microcon 10 filtrate of Vf ; Vmr, Microcon 10 retentate of Vf; Vb, Vf boiled for 10 minutes. Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The blot shown is representative of two experiments.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: Western blot analysis of ferritin H- and L-chains in the lysates of LEC treated with differentially processed fractions of vitreous humor. LEC were treated for 24 hours without (C) or with 33% of vitreous humor (V) and with equivalent amount of differentially processed vitreous fractions, in DMEM containing 10% FBS. Vf, vitreous humor filtered through glass wool; Va, acetone precipitate of Vf dissolved in complete DMEM; Vmf, Microcon 10 filtrate of Vf ; Vmr, Microcon 10 retentate of Vf; Vb, Vf boiled for 10 minutes. Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The blot shown is representative of two experiments.

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Western Blot, SDS Page, Purification, Imaging

    Effects of treatment of LEC with vitreous humor collected from dogs 7 to 10 years old with (Vcat) or without (V) age-related cataract on expression of ferritin chains and TfR1. LEC were treated with 33% vitreous humor in DMEM containing 10% FBS for 96 hours. Cells nontreated with vitreous humors were used as controls (C). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains, and purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 7; statistically different from corresponding V, * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Vitreous Humor Changes Expression of Iron-Handling Proteins in Lens Epithelial Cells

    doi: 10.1167/iovs.16-20610

    Figure Lengend Snippet: Effects of treatment of LEC with vitreous humor collected from dogs 7 to 10 years old with (Vcat) or without (V) age-related cataract on expression of ferritin chains and TfR1. LEC were treated with 33% vitreous humor in DMEM containing 10% FBS for 96 hours. Cells nontreated with vitreous humors were used as controls (C). Cell lysates containing 35 to 50 μg protein were separated by 12% SDS-PAGE, under reducing conditions, using a Tris/Tricine buffer system and transferred to nitrocellulose membranes. Purified canine heart ferritin was used as standard (St) for ferritin chains, and purified human placenta TfR1 was used as a standard (St) for canine TfR1. After Western transfer to a nitrocellulose membrane, ferritin chains were immunodetected with canine chain–specific custom-made antibodies. TfR1 was immunodetected with monoclonal mouse anti-human TfR1 antibodies. Blots were reprobed with HRP-goat anti-human β-actin as a loading control and evaluated with ChemiDoc MP Imaging System. The significance of differences was determined by paired t -test. Data represent the mean ± SEM; n = 7; statistically different from corresponding V, * P

    Article Snippet: Lens epithelial cells that grew out from capsule were dispersed with trypsin and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Rockville, MD, USA) with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen).

    Techniques: Expressing, SDS Page, Purification, Western Blot, Imaging

    Effect of FGF2 and/or TGFβ2 stimulation on activation of ERK1/2 pathway in MLECs. To evaluate the effect of FGF2 with/without TGFβ2 stimulation on phosphorylation of ERK1/2 in MLECs, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 10 or 60 min. Cell lysates were prepared, and Western blotting analysis was performed using anti‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab or anti‐phospho‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab. Data were from three experiments and were reported as means ± S.D.s.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Effect of FGF2 and/or TGFβ2 stimulation on activation of ERK1/2 pathway in MLECs. To evaluate the effect of FGF2 with/without TGFβ2 stimulation on phosphorylation of ERK1/2 in MLECs, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 10 or 60 min. Cell lysates were prepared, and Western blotting analysis was performed using anti‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab or anti‐phospho‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab. Data were from three experiments and were reported as means ± S.D.s.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Activation Assay, Western Blot

    Effect of MECK inhibitor (PD) on migration of MLECs treated with TGFβ2 and FGF2. MLECs were plated in 24‐well plates, pre‐coated with collagen type I, at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. After hydrogel removal to expose the cell‐free region, MLECs were treated with 0 or 10 ng/ml of TGFβ2 plus FGF2 with/without PD in DMEM containing 0.1% BSA for 24 hrs. Phase contrast micrographs were then taken with a digital camera. Data were from three experiments and were reported as means ± S.D.s. Scale bar, 180 μm.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Effect of MECK inhibitor (PD) on migration of MLECs treated with TGFβ2 and FGF2. MLECs were plated in 24‐well plates, pre‐coated with collagen type I, at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. After hydrogel removal to expose the cell‐free region, MLECs were treated with 0 or 10 ng/ml of TGFβ2 plus FGF2 with/without PD in DMEM containing 0.1% BSA for 24 hrs. Phase contrast micrographs were then taken with a digital camera. Data were from three experiments and were reported as means ± S.D.s. Scale bar, 180 μm.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Migration

    Migration of MLECs treated with/without TGFβ2 and/or FGF2. MLECs were plated in 24‐well plates pre‐coated with collagen type I, at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. Each plate contained 0.68 mm non‐toxic biocompatible hydrogel spot (Radius™ Gel) where cells cannot attach. After hydrogel removal to expose the cell‐free region, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 24 hrs. Phase contrast micrographs were then taken with a digital camera. Data shown are representative of three experiments. The cell‐free area was analysed using MultiGauge Software (Fuji Film, Tokyo, Japan). Data were from three experiments and were reported as means ± S.D.s. Scale bar, 180 μm.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Migration of MLECs treated with/without TGFβ2 and/or FGF2. MLECs were plated in 24‐well plates pre‐coated with collagen type I, at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. Each plate contained 0.68 mm non‐toxic biocompatible hydrogel spot (Radius™ Gel) where cells cannot attach. After hydrogel removal to expose the cell‐free region, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 24 hrs. Phase contrast micrographs were then taken with a digital camera. Data shown are representative of three experiments. The cell‐free area was analysed using MultiGauge Software (Fuji Film, Tokyo, Japan). Data were from three experiments and were reported as means ± S.D.s. Scale bar, 180 μm.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Migration, Software

    Expression of Tpm1/2 and αSMA in response to TGFβ1 and TGFβ2 in MLECs. Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. LECs were treated with 10 ng/ml of TGFβ1 or TGFβ2 in DMEM containing 0.1% BSA for 2 days. ( A ) Cell lysates were prepared, and Western blotting analysis was performed. αSMA was used as the marker of EMT. GAPDH was used for control of protein concentration on Western blot analysis. Relative densities of Tpm1/2, αSMA and GAPDH were determined using the Image Quant LAS 4000 (GE Healthcare UK Ltd. Buckinghamshire, England). Data are representative of three experiments. ( B ) Total RNA was prepared, and real‐time PCR analysis was performed. 18s ribosomal RNA was used for control of cDNA concentration on real‐time PCR analysis. Relative quantity of Tpm1/2 was determined using Prism 7300 System SDS RQ Study Software (Applied Biosystems ® ). Data were from three experiments and were reported as means ± S.D.s.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Expression of Tpm1/2 and αSMA in response to TGFβ1 and TGFβ2 in MLECs. Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. LECs were treated with 10 ng/ml of TGFβ1 or TGFβ2 in DMEM containing 0.1% BSA for 2 days. ( A ) Cell lysates were prepared, and Western blotting analysis was performed. αSMA was used as the marker of EMT. GAPDH was used for control of protein concentration on Western blot analysis. Relative densities of Tpm1/2, αSMA and GAPDH were determined using the Image Quant LAS 4000 (GE Healthcare UK Ltd. Buckinghamshire, England). Data are representative of three experiments. ( B ) Total RNA was prepared, and real‐time PCR analysis was performed. 18s ribosomal RNA was used for control of cDNA concentration on real‐time PCR analysis. Relative quantity of Tpm1/2 was determined using Prism 7300 System SDS RQ Study Software (Applied Biosystems ® ). Data were from three experiments and were reported as means ± S.D.s.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Expressing, Cell Culture, Western Blot, Marker, Protein Concentration, Real-time Polymerase Chain Reaction, Concentration Assay, Software

    Expression of Tpm1 and αSMA proteins in MLECs and HLECs stimulated with FGF2 and TGFβ2. Cultured MLECs ( A ) and HLECs ( B ) were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. ( A ) MLECs were treated with 0 or 10 ng/ml of TGFβ2 and FGF2 in DMEM containing 0.1% BSA for 2 and 4 days. αSMA was used for marker of EMT. ( B ) HLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 4 days. A and B : Cell lysates were prepared, and Western blotting analysis was performed, with GAPDH used for control of protein concentration. Data were from three experiments and were reported as means ± S.D.s.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Expression of Tpm1 and αSMA proteins in MLECs and HLECs stimulated with FGF2 and TGFβ2. Cultured MLECs ( A ) and HLECs ( B ) were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. ( A ) MLECs were treated with 0 or 10 ng/ml of TGFβ2 and FGF2 in DMEM containing 0.1% BSA for 2 and 4 days. αSMA was used for marker of EMT. ( B ) HLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 4 days. A and B : Cell lysates were prepared, and Western blotting analysis was performed, with GAPDH used for control of protein concentration. Data were from three experiments and were reported as means ± S.D.s.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Expressing, Cell Culture, Marker, Western Blot, Protein Concentration

    Expression of Tpm1 and αSMA in MLECs in response to FGF2. Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. ( A ) MLECs were treated with 0 or 10 ng/ml of FGF2 in DMEM containing 0.1% BSA for 2 days. αSMA was used as the marker of EMT. ( B ) MLECs were treated with 0, 0.01, 0.1, 1.0 or 10 ng/ml of FGF2 in DMEM containing 0.1% BSA for 2 days. A and B : Cell lysates were prepared, and Western blotting analysis was performed, with GAPDH used for control of protein concentration. Data were from three experiments and were reported as means ± S.D.s.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Expression of Tpm1 and αSMA in MLECs in response to FGF2. Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. ( A ) MLECs were treated with 0 or 10 ng/ml of FGF2 in DMEM containing 0.1% BSA for 2 days. αSMA was used as the marker of EMT. ( B ) MLECs were treated with 0, 0.01, 0.1, 1.0 or 10 ng/ml of FGF2 in DMEM containing 0.1% BSA for 2 days. A and B : Cell lysates were prepared, and Western blotting analysis was performed, with GAPDH used for control of protein concentration. Data were from three experiments and were reported as means ± S.D.s.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Expressing, Cell Culture, Marker, Western Blot, Protein Concentration

    Effect of FGFR antagonist (SU) and MECK inhibitor (PD) on FGF2‐induced activation of ERK pathway. MECK inhibitor (PD) to block ERK pathway and FGFR antagonist (SU) to inhibit FGF2 stimulation were used. To evaluate the effect of FGF2 with/without TGFβ2 stimulation on phosphorylation of ERK1/2, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA with/without SU or PD for 10 min or 24 hrs. Cell lysates were prepared, and Western blotting analysis was performed using anti‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab or anti‐phospho‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab. Data were from three experiments and were reported as means ± S.D.s.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Effect of FGFR antagonist (SU) and MECK inhibitor (PD) on FGF2‐induced activation of ERK pathway. MECK inhibitor (PD) to block ERK pathway and FGFR antagonist (SU) to inhibit FGF2 stimulation were used. To evaluate the effect of FGF2 with/without TGFβ2 stimulation on phosphorylation of ERK1/2, MLECs were treated with 0 or 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA with/without SU or PD for 10 min or 24 hrs. Cell lysates were prepared, and Western blotting analysis was performed using anti‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab or anti‐phospho‐rabbit p44/42 MAPK (Erk1/2) monoclonal Ab. Data were from three experiments and were reported as means ± S.D.s.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Activation Assay, Blocking Assay, Western Blot

    TGFβ2‐ and/or FGF2‐mediated phenotypic changes of LECs in vitro . Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. LECs were treated with 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 2 days. Phase contrast photomicrographs were taken with a digital camera. Data were from three experiments. Scale bar, 90 μm.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: TGFβ2‐ and/or FGF2‐mediated phenotypic changes of LECs in vitro . Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. LECs were treated with 10 ng/ml of TGFβ2 and/or FGF2 in DMEM containing 0.1% BSA for 2 days. Phase contrast photomicrographs were taken with a digital camera. Data were from three experiments. Scale bar, 90 μm.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: In Vitro, Cell Culture

    Effect of MECK inhibitor (PD) and FGF2 antagonist (SU) on repression of Tpm1 and αSMA expression in response to FGF2. Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. MLECs were treated with 0 or 10 ng/ml of TGFβ2 plus FGF2 in DMEM containing 0.1% BSA with/without SU or PD for 60 min and 24 hrs. Cell lysates were prepared, and Western blotting analysis was performed using anti‐Tpm1/2 Ab and anti αSMA Ab, with GAPDH used for control of protein concentration. Data were from three experiments and were reported as means ± S.D.s.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

    doi: 10.1111/jcmm.13030

    Figure Lengend Snippet: Effect of MECK inhibitor (PD) and FGF2 antagonist (SU) on repression of Tpm1 and αSMA expression in response to FGF2. Cultured MLECs were plated in 35 mm dishes at a density of 1 × 10 5 in DMEM with 10%FBS for 24 hrs. MLECs were treated with 0 or 10 ng/ml of TGFβ2 plus FGF2 in DMEM containing 0.1% BSA with/without SU or PD for 60 min and 24 hrs. Cell lysates were prepared, and Western blotting analysis was performed using anti‐Tpm1/2 Ab and anti αSMA Ab, with GAPDH used for control of protein concentration. Data were from three experiments and were reported as means ± S.D.s.

    Article Snippet: Cells growing in DMEM containing 0.1% bovine serum albumin (BSA) (WAKO) in the presence or absence of various test growth regulators received 0.001–10.0 ng/ml FGF‐2 or 0–10 ng/ml TGF‐β2 every other day for up to 4 days.

    Techniques: Expressing, Cell Culture, Western Blot, Protein Concentration

    CHX partially inhibited complement-mediated ApoE accumulation. RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant ( A ) and a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant ( B ) were preincubated with CHX at 20 μg/mL for 1.5 hours, primed with or without S58 (1.2 mg/mL) for 30 minutes, and then treated with either 6% C1q-Dep or HiC1q-Dep for 5 hours. CHX was continuously present when cells were primed with S58 and incubated with complement serum. Total proteins (15 μg) were separated by SDS-PAGE for Western blot. The quantity of ApoE relative to GAPDH shown in ( A , B ) was determined by densitometry (labeling 1–5 represents five treatment groups). * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: CHX partially inhibited complement-mediated ApoE accumulation. RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant ( A ) and a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant ( B ) were preincubated with CHX at 20 μg/mL for 1.5 hours, primed with or without S58 (1.2 mg/mL) for 30 minutes, and then treated with either 6% C1q-Dep or HiC1q-Dep for 5 hours. CHX was continuously present when cells were primed with S58 and incubated with complement serum. Total proteins (15 μg) were separated by SDS-PAGE for Western blot. The quantity of ApoE relative to GAPDH shown in ( A , B ) was determined by densitometry (labeling 1–5 represents five treatment groups). * P

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Variant Assay, Incubation, SDS Page, Western Blot, Labeling

    Detection of cleaved recombinant ApoE human protein in serum-treated cells. RPE cells from a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with 6% either C1q-Dep or HiC1q-Dep for 5 hours with or without recombinant ApoE human protein with N-TRX.His tags (4 μg/mL). Total proteins (15 μg) were separated by SDS-PAGE. Data are representative of four separate experiments in two donors with similar results. *S58 IgG heavy chain.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Detection of cleaved recombinant ApoE human protein in serum-treated cells. RPE cells from a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with 6% either C1q-Dep or HiC1q-Dep for 5 hours with or without recombinant ApoE human protein with N-TRX.His tags (4 μg/mL). Total proteins (15 μg) were separated by SDS-PAGE. Data are representative of four separate experiments in two donors with similar results. *S58 IgG heavy chain.

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Recombinant, Variant Assay, SDS Page

    Repetitive complement challenge enhanced cell-associated ApoE accumulation. RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep for 24 or 48 hours. For the second and third complement challenges, cells were washed once, reprimed with or without S58, and then incubated with 6% C1q-Dep or HiC1q-Dep for 48 hours. Repetitive challenge was performed every other day for 1 week (three challenges). Total proteins (15 μg) were separated by SDS-PAGE. The relative quantity of ApoE protein normalized to GAPDH is shown below each lane . Blot images are representative of seven separate experiments in three donors with similar results. The bar graph depicts pooled data from two separate experiments in which cells from a single donor were treated under identical conditions.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Repetitive complement challenge enhanced cell-associated ApoE accumulation. RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep for 24 or 48 hours. For the second and third complement challenges, cells were washed once, reprimed with or without S58, and then incubated with 6% C1q-Dep or HiC1q-Dep for 48 hours. Repetitive challenge was performed every other day for 1 week (three challenges). Total proteins (15 μg) were separated by SDS-PAGE. The relative quantity of ApoE protein normalized to GAPDH is shown below each lane . Blot images are representative of seven separate experiments in three donors with similar results. The bar graph depicts pooled data from two separate experiments in which cells from a single donor were treated under identical conditions.

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Variant Assay, Incubation, SDS Page

    Cell suspension model confirmed cell-associated ApoE accumulation. ( A ) Suspended RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were incubated with either normal sheep IgG (1.2 mg/mL) or S58 (1.2 mg/mL) for 30 minutes and then treated with 6% C1q-Dep for 4.5 hours at 37°C in a shaking incubator followed by cell lysis and SDS-PAGE analysis of total proteins (30 μg). ( B ) Quantitation of cell-associated ApoE relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) shown in ( A ) was determined by densitometry. * P = 0.02 versus sheep IgG+C1q-Dep. Data are representative of two separate experiments in two donors with similar results.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Cell suspension model confirmed cell-associated ApoE accumulation. ( A ) Suspended RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were incubated with either normal sheep IgG (1.2 mg/mL) or S58 (1.2 mg/mL) for 30 minutes and then treated with 6% C1q-Dep for 4.5 hours at 37°C in a shaking incubator followed by cell lysis and SDS-PAGE analysis of total proteins (30 μg). ( B ) Quantitation of cell-associated ApoE relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) shown in ( A ) was determined by densitometry. * P = 0.02 versus sheep IgG+C1q-Dep. Data are representative of two separate experiments in two donors with similar results.

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Variant Assay, Incubation, Lysis, SDS Page, Quantitation Assay

    Repetitive sublytic complement challenge caused ApoE deposition in ECM. RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were primed with either normal sheep IgG (0.4 mg/mL) or S58 (0.4 mg/mL) for 30 minutes and then treated with 6% C1q-Dep or HiC1q-Dep for 48 hours. Repetitive sublytic challenge was produced as described in the Methods section. RPE cells were removed after the third complement challenge by incubating cells with 0.02 N ammonium hydroxide (NH 4 OH). ( A ) Similar cell morphology was observed across treatment groups prior to adding NH 4 OH and complete cell removal after adding NH 4 OH. ( B ) Equal lysate volumes were separated by SDS-PAGE and probed with ApoE. ( C ) The quantity of ApoE shown in ( B ) was determined by densitometry and normalized to protein concentration. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Repetitive sublytic complement challenge caused ApoE deposition in ECM. RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were primed with either normal sheep IgG (0.4 mg/mL) or S58 (0.4 mg/mL) for 30 minutes and then treated with 6% C1q-Dep or HiC1q-Dep for 48 hours. Repetitive sublytic challenge was produced as described in the Methods section. RPE cells were removed after the third complement challenge by incubating cells with 0.02 N ammonium hydroxide (NH 4 OH). ( A ) Similar cell morphology was observed across treatment groups prior to adding NH 4 OH and complete cell removal after adding NH 4 OH. ( B ) Equal lysate volumes were separated by SDS-PAGE and probed with ApoE. ( C ) The quantity of ApoE shown in ( B ) was determined by densitometry and normalized to protein concentration. * P

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Variant Assay, Produced, SDS Page, Protein Concentration

    ApoE was colocalized with MAC on complement-activated RPE cell culture and drusen. ( A ) RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were primed with S58 (1.2 mg/mL) for 30 minutes and then treated with 6% C1q-Dep for 5 hours. Cells were fixed in 4% PFA for 15 minutes and costained with rabbit anti-human ApoE antibody ( green ) and mouse anti-human C5b-9 (aE11) antibody ( red ). Blue stain corresponds to DAPI-stained nuclei. MAC and ApoE were colocalized frequently in the cells. Data are representative of three separate experiments in three donors with similar results. ( B ) Confocal microscopic colocalization ( yellow ) of MAC ( green ) and ApoE ( red ) in drusen from a 72-year-old donor. Similar results were observed in 94-year-old donor eye (not shown). ( C ) Primary antibodies were omitted in section of a 72-year-old donor. Scale bar : 50 μm.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: ApoE was colocalized with MAC on complement-activated RPE cell culture and drusen. ( A ) RPE cells from a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were primed with S58 (1.2 mg/mL) for 30 minutes and then treated with 6% C1q-Dep for 5 hours. Cells were fixed in 4% PFA for 15 minutes and costained with rabbit anti-human ApoE antibody ( green ) and mouse anti-human C5b-9 (aE11) antibody ( red ). Blue stain corresponds to DAPI-stained nuclei. MAC and ApoE were colocalized frequently in the cells. Data are representative of three separate experiments in three donors with similar results. ( B ) Confocal microscopic colocalization ( yellow ) of MAC ( green ) and ApoE ( red ) in drusen from a 72-year-old donor. Similar results were observed in 94-year-old donor eye (not shown). ( C ) Primary antibodies were omitted in section of a 72-year-old donor. Scale bar : 50 μm.

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Cell Culture, Variant Assay, Staining

    Cell-associated ApoE accumulation was dependent on MAC deposition. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with 6% serum for 30 minutes ( A ), 6 hours ( B , C ), or 5 hours ( D , E ). ( A ) Induction of MAC accumulation on RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant. After serum treatments in the presence or absence of anti-C5 antibody (10 μg/mL), cells were fixed in 4% PFA for 15 minutes and stained with mouse anti-human C5b-9 (aE11) antibody. Data are representative of two separate experiments in two donors with similar results. Red stain indicates MAC deposition. Blue stain corresponds to 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar : 100 μm. ( B ) Accumulation of ApoE was prevented when C5 was blocked by anti-C5 antibody. Total proteins (15 μg) obtained from RPE cells of a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant were separated by SDS-PAGE 6 hours post complement challenge in the presence or absence of anti-C5 antibody (10 μg/mL). ( C ) The quantity of ApoE relative to GAPDH shown in ( B ) was determined by densitometry. * P = 0.001 vs. C1q-Dep and C5 Ab+C1q-Dep. ** P = 0.002 vs. S58+C1q-Dep. Data are representative of three separate experiments in three donors with similar results. ( D ) Accumulation of ApoE was blocked by absence of C6. Total proteins (30 μg) obtained from RPE cells of a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were separated by SDS-PAGE after treatment with C6-Dep in the presence or absence of C6 protein at 7.3 or 65 μg/mL. ( E ) The quantity of ApoE relative to GAPDH shown in ( D ) was determined by densitometry. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Cell-associated ApoE accumulation was dependent on MAC deposition. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with 6% serum for 30 minutes ( A ), 6 hours ( B , C ), or 5 hours ( D , E ). ( A ) Induction of MAC accumulation on RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant. After serum treatments in the presence or absence of anti-C5 antibody (10 μg/mL), cells were fixed in 4% PFA for 15 minutes and stained with mouse anti-human C5b-9 (aE11) antibody. Data are representative of two separate experiments in two donors with similar results. Red stain indicates MAC deposition. Blue stain corresponds to 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar : 100 μm. ( B ) Accumulation of ApoE was prevented when C5 was blocked by anti-C5 antibody. Total proteins (15 μg) obtained from RPE cells of a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant were separated by SDS-PAGE 6 hours post complement challenge in the presence or absence of anti-C5 antibody (10 μg/mL). ( C ) The quantity of ApoE relative to GAPDH shown in ( B ) was determined by densitometry. * P = 0.001 vs. C1q-Dep and C5 Ab+C1q-Dep. ** P = 0.002 vs. S58+C1q-Dep. Data are representative of three separate experiments in three donors with similar results. ( D ) Accumulation of ApoE was blocked by absence of C6. Total proteins (30 μg) obtained from RPE cells of a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were separated by SDS-PAGE after treatment with C6-Dep in the presence or absence of C6 protein at 7.3 or 65 μg/mL. ( E ) The quantity of ApoE relative to GAPDH shown in ( D ) was determined by densitometry. * P

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Variant Assay, Staining, SDS Page

    Western blot confirmed that complement activation increases cell-associated ApoE protein determined by LC-MS/MS analysis. RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant were primed with S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep for 6 hours. Total proteins (30 μg) were separated by SDS-PAGE. Lanes 1 and 3 used the same total protein samples as those in experiment 1 in the Table . Lanes 2 and 4 used the same total protein samples as those in experiment 2 in the Table .

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Western blot confirmed that complement activation increases cell-associated ApoE protein determined by LC-MS/MS analysis. RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant were primed with S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep for 6 hours. Total proteins (30 μg) were separated by SDS-PAGE. Lanes 1 and 3 used the same total protein samples as those in experiment 1 in the Table . Lanes 2 and 4 used the same total protein samples as those in experiment 2 in the Table .

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Western Blot, Activation Assay, Liquid Chromatography, Mass Spectrometry, Variant Assay, SDS Page

    Time-dependent ApoE accumulation in complement-treated cells. RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant were primed with or without S58 (0.8 or 1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep (control) for 1 to 6 hours ( A ) and 6 to 24 hours ( B ) Total proteins (15 μg) were separated by SDS-PAGE. The quantity of ApoE protein and ApoA1 protein is shown below each lane .

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Time-dependent ApoE accumulation in complement-treated cells. RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant were primed with or without S58 (0.8 or 1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep (control) for 1 to 6 hours ( A ) and 6 to 24 hours ( B ) Total proteins (15 μg) were separated by SDS-PAGE. The quantity of ApoE protein and ApoA1 protein is shown below each lane .

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Variant Assay, SDS Page

    Steady-state ApoE mRNA levels were not affected by complement challenge. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep for various times. RNA in triplicate samples was extracted, reverse transcribed to cDNA, amplified with ApoE- ( A ) or IL-6- ( B ) specific primer pairs, and quantified by qPCR. Data are representative of four separate experiments in a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant with similar results. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: Steady-state ApoE mRNA levels were not affected by complement challenge. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or HiC1q-Dep for various times. RNA in triplicate samples was extracted, reverse transcribed to cDNA, amplified with ApoE- ( A ) or IL-6- ( B ) specific primer pairs, and quantified by qPCR. Data are representative of four separate experiments in a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant with similar results. * P

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Amplification, Real-time Polymerase Chain Reaction, Variant Assay

    ApoE protein in conditioned media was elevated in response to complement challenge. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or 6% HiC1q-Dep for 6 hours. The medium was then exchanged with serum-free medium. After media exchange, cultures were incubated for either 1, 24, 48, or 72 hours and levels of soluble ApoE in the media were determined. Total cellular lysate proteins (15 μg) and concentrated conditioned media (30 μL) were separated by SDS-PAGE. The data are representative of two separate experiments in a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant with similar results.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

    doi: 10.1167/iovs.16-20083

    Figure Lengend Snippet: ApoE protein in conditioned media was elevated in response to complement challenge. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with either 6% C1q-Dep or 6% HiC1q-Dep for 6 hours. The medium was then exchanged with serum-free medium. After media exchange, cultures were incubated for either 1, 24, 48, or 72 hours and levels of soluble ApoE in the media were determined. Total cellular lysate proteins (15 μg) and concentrated conditioned media (30 μL) were separated by SDS-PAGE. The data are representative of two separate experiments in a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant with similar results.

    Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

    Techniques: Incubation, SDS Page, Variant Assay

    HPV18 requires EGFR activation to maintain cyclin B1 expression in differentiating cells (A) Schematic showing the EGFR/ERK signalling pathway and the targets of the siRNA and inhibitors used in this study. Mock treatedkeratinocytes were differentiated in high calcium media and lysed after 48 hours. In parallel, keratinocytes were treated with (B) scramble or EGFR specific siRNA, (C) an EGFR kinase inhibitor (2 μM PD153035), or (D) a Mek1/2 kinase inhibitor (20 μM UO126) during differentiation. All samples were analysed for cyclin B1 and phosphorylated ERK1/2 expression. GAPDH served as a loading control. Representative blots are shown from at least three independent biological repeats from two donor lines.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 requires EGFR activation to maintain cyclin B1 expression in differentiating cells (A) Schematic showing the EGFR/ERK signalling pathway and the targets of the siRNA and inhibitors used in this study. Mock treatedkeratinocytes were differentiated in high calcium media and lysed after 48 hours. In parallel, keratinocytes were treated with (B) scramble or EGFR specific siRNA, (C) an EGFR kinase inhibitor (2 μM PD153035), or (D) a Mek1/2 kinase inhibitor (20 μM UO126) during differentiation. All samples were analysed for cyclin B1 and phosphorylated ERK1/2 expression. GAPDH served as a loading control. Representative blots are shown from at least three independent biological repeats from two donor lines.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Activation Assay, Expressing

    HPV18 E5 inhibits downstream targets of the KGFR signalling pathway (A) Schematic showing the KGFR downstream signalling pathway including the KGFR substrate FRS2a and downstream kinases AKT and GSK3β. (B) Lysates of NHK, WT and E5KO keratinocytes subjected to high calcium differentiation were analysed for p-AKT, total AKT and p-GSK3β. GAPDH served as a loading control. Western blots are representative of three independent experiments in two donor lines

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 E5 inhibits downstream targets of the KGFR signalling pathway (A) Schematic showing the KGFR downstream signalling pathway including the KGFR substrate FRS2a and downstream kinases AKT and GSK3β. (B) Lysates of NHK, WT and E5KO keratinocytes subjected to high calcium differentiation were analysed for p-AKT, total AKT and p-GSK3β. GAPDH served as a loading control. Western blots are representative of three independent experiments in two donor lines

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Western Blot

    Schematic model depicting the proposed role of EGFR signalling in the E5-mediated manipulation of proliferation and differentiation pathways during the virus life cycle E5 expression results in enhanced EGFR surface expression and activation, through a process that might require endosome recycling (1). This increases ERK/MAPK activity (2), resulting in activation of substrates, which include cell cycle associated proteins e.g. cyclin B (3). EGFR can suppress keratinocyte differentiation, by inhibiting a number of targets including the KGFR pathway. KGFR transcription is suppressed in E5 expressing cells (4) and KGFR signalling is markedly reduced (5). As a consequence, targets of KGFR including Akt are suppressed, culminating in loss of expression of a number of spinous associated differentiation markers e.g. cytokeratin 1 (K1) (6). As a result keratinocyte differentiation is impaired in cells expressing E5.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: Schematic model depicting the proposed role of EGFR signalling in the E5-mediated manipulation of proliferation and differentiation pathways during the virus life cycle E5 expression results in enhanced EGFR surface expression and activation, through a process that might require endosome recycling (1). This increases ERK/MAPK activity (2), resulting in activation of substrates, which include cell cycle associated proteins e.g. cyclin B (3). EGFR can suppress keratinocyte differentiation, by inhibiting a number of targets including the KGFR pathway. KGFR transcription is suppressed in E5 expressing cells (4) and KGFR signalling is markedly reduced (5). As a consequence, targets of KGFR including Akt are suppressed, culminating in loss of expression of a number of spinous associated differentiation markers e.g. cytokeratin 1 (K1) (6). As a result keratinocyte differentiation is impaired in cells expressing E5.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Expressing, Activation Assay, Activity Assay

    HPV18 E5 inhibits the KGFR signalling pathway (A) Organotypic raft cultures stained for KGFR expression (red) and nuclei stained with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Images are representative of staining from two donors. (B) Histogram analysis of the staining intensity from WT and E5KO rafts. Data was derived from 5 fields of view from three independent experiments, from two donors. (C) Graph showing relative KGFR mRNA expression in differentiated WT and E5KO keratinocytes after 96 hours incubation in high calcium media. Results were corrected for expression of an U6 loading control. The data is shown as a mean and standard deviation from three independent biological repeats from two donors. Significance as determined by Student’s t-test is shown as ** = p

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 E5 inhibits the KGFR signalling pathway (A) Organotypic raft cultures stained for KGFR expression (red) and nuclei stained with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Images are representative of staining from two donors. (B) Histogram analysis of the staining intensity from WT and E5KO rafts. Data was derived from 5 fields of view from three independent experiments, from two donors. (C) Graph showing relative KGFR mRNA expression in differentiated WT and E5KO keratinocytes after 96 hours incubation in high calcium media. Results were corrected for expression of an U6 loading control. The data is shown as a mean and standard deviation from three independent biological repeats from two donors. Significance as determined by Student’s t-test is shown as ** = p

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Staining, Expressing, Derivative Assay, Incubation, Standard Deviation

    Pharmacological inhibition of KGFR activity compensates for loss of E5 and impairs keratinocyte differentiation (A) Schematic showing the KGFR signalling pathway and target of the pharmacological inhibitor. (B) Control (vehicle only) keratinocytes were differentiated in high calcium media and lysed after 48 hours. Parallel cultures of E5KO keratinocytes were treated with 30 nM of a KGFR kinase inhibitor (PD173074). Lysates were analysed for the phosphorylated forms of FRS2α, AKT, GSK3α/β, cytokeratin 1 (K1) and cyclin B1 by immunoblot.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: Pharmacological inhibition of KGFR activity compensates for loss of E5 and impairs keratinocyte differentiation (A) Schematic showing the KGFR signalling pathway and target of the pharmacological inhibitor. (B) Control (vehicle only) keratinocytes were differentiated in high calcium media and lysed after 48 hours. Parallel cultures of E5KO keratinocytes were treated with 30 nM of a KGFR kinase inhibitor (PD173074). Lysates were analysed for the phosphorylated forms of FRS2α, AKT, GSK3α/β, cytokeratin 1 (K1) and cyclin B1 by immunoblot.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Inhibition, Activity Assay

    HPV18 E5 function is not necessary for genome amplification and late protein expression in differentiating keratinocytes (A) Organotypic raft cultures were probed with a biotin-conjugated HPV DNA specific for the high-risk HPV types to visualise genome amplification. Arrows indicate examples of CISH positive nuclei. Graph represents the mean (± standard deviation) of CISH positive nuclei per field of view (15 fields of view of each raft) for two separate donors. (B and C) Organotypic rafts stained for E4 (red) and L1 (green), nuclei were visualised with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Images are representative of staining from two donors and three independent experiments.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 E5 function is not necessary for genome amplification and late protein expression in differentiating keratinocytes (A) Organotypic raft cultures were probed with a biotin-conjugated HPV DNA specific for the high-risk HPV types to visualise genome amplification. Arrows indicate examples of CISH positive nuclei. Graph represents the mean (± standard deviation) of CISH positive nuclei per field of view (15 fields of view of each raft) for two separate donors. (B and C) Organotypic rafts stained for E4 (red) and L1 (green), nuclei were visualised with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Images are representative of staining from two donors and three independent experiments.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Amplification, Expressing, Chromogenic In Situ Hybridization, Standard Deviation, Staining

    HPV18 E5 maintains EGFR expression in differentiating keratinocytes (A) Organotypic rafts stained for EGFR (green), with nuclei stained with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Images are representative of staining from two donors. (B) Histogram analysis of the staining intensity from WT and E5KO rafts. Data was derived from 5 fields of view from three independent experiments and two donors. (C) Lysates of keratinocytes differentiated in high calcium media analysed for total EGFR expression by immunoblotting. GAPDH served as a loading control. Western blots are representative of three independent experiments in two donor lines.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 E5 maintains EGFR expression in differentiating keratinocytes (A) Organotypic rafts stained for EGFR (green), with nuclei stained with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Images are representative of staining from two donors. (B) Histogram analysis of the staining intensity from WT and E5KO rafts. Data was derived from 5 fields of view from three independent experiments and two donors. (C) Lysates of keratinocytes differentiated in high calcium media analysed for total EGFR expression by immunoblotting. GAPDH served as a loading control. Western blots are representative of three independent experiments in two donor lines.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Expressing, Staining, Derivative Assay, Western Blot

    HPV18 E5 impairs keratinocyte differentiation Lysates of NHK, WT and E5KO keratinocytes subjected to high calcium differentiation were analysed for involucrin and cytokeratin 1 (K1) expression. GAPDH served as a loading control. Western blots are representative of three independent experiments in two donor lines.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 E5 impairs keratinocyte differentiation Lysates of NHK, WT and E5KO keratinocytes subjected to high calcium differentiation were analysed for involucrin and cytokeratin 1 (K1) expression. GAPDH served as a loading control. Western blots are representative of three independent experiments in two donor lines.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Expressing, Western Blot

    HPV18 E5 contributes towards unscheduled host cell DNA synthesis upon keratinocyte differentiation (A) Southern analysis of equal amounts of total DNA extracted from NHK transfected with wild-type (WT) or mutant (E5KO) genomes in two different donors. DNA was digested with Dpn I (digests input DNA) together with Bgl II which does not digest HPV18 DNA or Eco RI which linearizes HPV18 genomes. (B) Detection of E6 and E7 proteins in equal amounts of Triton-X100 detergent soluble protein lysates prepared from cells differentiated in high-calcium monolayer cultures. Levels of GAPDH were used as a loading control. Densitometry analysis of protein bands was performed using ImageJ software. (C) Organotypic rafts were incubated with BrdU to identify nuclei positive for cellular DNA synthesis. BrdU positive cells were visualised with an anti-BrdU antibody (red) and nuclei visualised with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Organotypic raft cultures were also stained with haematoxylin and eosin to observe the gross morphology of the epithelium. (D) Graphs showing the percentage of BrdU positive nuclei in basal and lower suprabasal and upper suprabasal layers. The data, shown as a mean with standard deviation, were derived from 15 fields of view of each raft and from 3 independent experiments from two donors cell lines. Significance as determined by Student’s t-test is shown as ** = p

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: HPV18 E5 contributes towards unscheduled host cell DNA synthesis upon keratinocyte differentiation (A) Southern analysis of equal amounts of total DNA extracted from NHK transfected with wild-type (WT) or mutant (E5KO) genomes in two different donors. DNA was digested with Dpn I (digests input DNA) together with Bgl II which does not digest HPV18 DNA or Eco RI which linearizes HPV18 genomes. (B) Detection of E6 and E7 proteins in equal amounts of Triton-X100 detergent soluble protein lysates prepared from cells differentiated in high-calcium monolayer cultures. Levels of GAPDH were used as a loading control. Densitometry analysis of protein bands was performed using ImageJ software. (C) Organotypic rafts were incubated with BrdU to identify nuclei positive for cellular DNA synthesis. BrdU positive cells were visualised with an anti-BrdU antibody (red) and nuclei visualised with DAPI (blue). White dotted lines indicate the basal cell layer. Red scale bar represents 20 μm. Organotypic raft cultures were also stained with haematoxylin and eosin to observe the gross morphology of the epithelium. (D) Graphs showing the percentage of BrdU positive nuclei in basal and lower suprabasal and upper suprabasal layers. The data, shown as a mean with standard deviation, were derived from 15 fields of view of each raft and from 3 independent experiments from two donors cell lines. Significance as determined by Student’s t-test is shown as ** = p

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: DNA Synthesis, Transfection, Mutagenesis, Software, Incubation, Staining, Standard Deviation, Derivative Assay

    AKT is essential for the modulation of keratinocyte differentiation by HPV18 (A) Schematic showing the KGFR signalling pathway and the effects of the dominant active (DA) and negative (DN) AKT. HPV18 genome containing keratinocytes were infected with empty retrovirus, or with retroviruses encoding DA AKT (B) or DN AKT (C) . Cells were differentiated in high calcium media for 48 hours prior to lysis and analysed for expression of cytokeratin 1 (K1) to assess differentiation. Expression of the exogenous AKT was confirmed using an antibody against the HA epitope and GAPDH served as a loading control.

    Journal: Oncotarget

    Article Title: Human papillomavirus type 18 E5 oncogene supports cell cycle progression and impairs epithelial differentiation by modulating growth factor receptor signalling during the virus life cycle

    doi: 10.18632/oncotarget.21658

    Figure Lengend Snippet: AKT is essential for the modulation of keratinocyte differentiation by HPV18 (A) Schematic showing the KGFR signalling pathway and the effects of the dominant active (DA) and negative (DN) AKT. HPV18 genome containing keratinocytes were infected with empty retrovirus, or with retroviruses encoding DA AKT (B) or DN AKT (C) . Cells were differentiated in high calcium media for 48 hours prior to lysis and analysed for expression of cytokeratin 1 (K1) to assess differentiation. Expression of the exogenous AKT was confirmed using an antibody against the HA epitope and GAPDH served as a loading control.

    Article Snippet: Media was changed to serum free keratinocyte media without supplements (SFM medium, Invitrogen) containing 1.8 mM calcium chloride.

    Techniques: Dominant Negative Mutation, Infection, Lysis, Expressing, Hemagglutination Assay

    The effects of collagen I gel concentrations on CD133 + cell migration in the 3D μ-slide assay. (A) After 24 hour culture of human UCB CD133 + cells in StemSpan medium and SCF, Flt-3 ligand, IL-6 and TPO, cells were suspended in 0.5 mg/ml, 1 mg/ml or 3 mg/ml collagen I gel solutions and seeded into the central chamber of the 3D chemotaxis μ-slides. A chemokine gradient was established using 1 μg/ml CXCL12. The cells were imaged over 22 h by timelapse microscopy migration and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug in for (a) accumulated distance, (b) velocity, (c) displacement of the center of mass and (d) forward migration index. Values are means ± SEM (n ≥ 3independent experiments). Variability amongst independent experiments was increased when 3 mg/ml collagen I gels were compared with 0.5 or 1 mg/ml collagen I gels. This is exemplified when comparing 1 mg/ml and 3 mg/ml collagen I gels respectively for the ranges in the accumulated distance (1012 μm to 1451 μm vs. 350 μm to 1467 μm respectively), velocity (0.77 to 1.16 vs. 0.27 to 1.18 μm/min respectively), displacement of center of mass (111.80 to 115.43 vs. 26.94 to 256.20 μm respectively) and forward migration index (0.100 to 0.110 vs. 0.060 to 0.220 respectively). (B) Microscopic images (× 4 magnification) of CD133 + cells in (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 3 mg/ml collagen 1 gels after seeding into the central chamber of the 3D chemotaxis μ-slide and demonstrating the lack of uniformity of collagen I fibers with 3 mg/ml collagen I gels.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: The effects of collagen I gel concentrations on CD133 + cell migration in the 3D μ-slide assay. (A) After 24 hour culture of human UCB CD133 + cells in StemSpan medium and SCF, Flt-3 ligand, IL-6 and TPO, cells were suspended in 0.5 mg/ml, 1 mg/ml or 3 mg/ml collagen I gel solutions and seeded into the central chamber of the 3D chemotaxis μ-slides. A chemokine gradient was established using 1 μg/ml CXCL12. The cells were imaged over 22 h by timelapse microscopy migration and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug in for (a) accumulated distance, (b) velocity, (c) displacement of the center of mass and (d) forward migration index. Values are means ± SEM (n ≥ 3independent experiments). Variability amongst independent experiments was increased when 3 mg/ml collagen I gels were compared with 0.5 or 1 mg/ml collagen I gels. This is exemplified when comparing 1 mg/ml and 3 mg/ml collagen I gels respectively for the ranges in the accumulated distance (1012 μm to 1451 μm vs. 350 μm to 1467 μm respectively), velocity (0.77 to 1.16 vs. 0.27 to 1.18 μm/min respectively), displacement of center of mass (111.80 to 115.43 vs. 26.94 to 256.20 μm respectively) and forward migration index (0.100 to 0.110 vs. 0.060 to 0.220 respectively). (B) Microscopic images (× 4 magnification) of CD133 + cells in (a) 0.5 mg/ml, (b) 1 mg/ml and (c) 3 mg/ml collagen 1 gels after seeding into the central chamber of the 3D chemotaxis μ-slide and demonstrating the lack of uniformity of collagen I fibers with 3 mg/ml collagen I gels.

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Migration, Chemotaxis Assay, Microscopy, Software

    Expression of CXCR4 on human cord blood CD133 + cells. (A (a)) Representative FACS dot plots of purified human UCB CD133 + cells (averaging 94 + 2% purity for the optimization and validation experiments) showing dual staining for respective isotype controls mIgG2a-APC vs. mIgG2b-PE or CD133-PE and CD34-APC antibodies after 24 hour culture in StemSpan serum free medium with SCF, Flt-3 ligand, IL-6 and TPO and hence at the commencement of the migration assays. (A (b to e)) Representative FACS histograms of cells from (A (a)) after mIgG2b-PE and CD133-PE or mIgG2a-APC and CXCR4-APC staining. (B) Median fluorescence intensities (MFIs) of n = 7 CD133 + cell preparations stained with the isotype control or the CXCR4 antibody with individual experiments represented by scatter plots and the median value being represented by the horizontal bar. For CXCR4 staining, MFIs averaged (mean ± SEM) 726.4 ± 153.3 compared to 38.0 + 8.5 for the negative isotype control.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: Expression of CXCR4 on human cord blood CD133 + cells. (A (a)) Representative FACS dot plots of purified human UCB CD133 + cells (averaging 94 + 2% purity for the optimization and validation experiments) showing dual staining for respective isotype controls mIgG2a-APC vs. mIgG2b-PE or CD133-PE and CD34-APC antibodies after 24 hour culture in StemSpan serum free medium with SCF, Flt-3 ligand, IL-6 and TPO and hence at the commencement of the migration assays. (A (b to e)) Representative FACS histograms of cells from (A (a)) after mIgG2b-PE and CD133-PE or mIgG2a-APC and CXCR4-APC staining. (B) Median fluorescence intensities (MFIs) of n = 7 CD133 + cell preparations stained with the isotype control or the CXCR4 antibody with individual experiments represented by scatter plots and the median value being represented by the horizontal bar. For CXCR4 staining, MFIs averaged (mean ± SEM) 726.4 ± 153.3 compared to 38.0 + 8.5 for the negative isotype control.

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Expressing, FACS, Purification, Staining, Migration, Fluorescence

    Comparison of chemotactic and chemokinetic responses of pooled versus non-pooled human cord blood CD133 + cells. Human CD133 + cells were isolated from umbilical cord blood and cultured as individual units (non-pooled; n = 7) or as pooled units (pooled; n = 8) in StemSpan medium containing SCF, Flt-3 ligand, IL-6 and TPO for 24 h before encapsulation into 1 mg/ml collagen I gel and seeding into the central chamber of a 3D chemotaxis μ-slide. Cell migration was tracked using timelapse microscopy and Image J software over 22 h in the presence of 1 μg/ml CXCL12. The chemotaxis and migration tool plug in was used to quantify (a) the percentage of cells migrating, (b) the displacement of the center of mass, (c) forward migration index, (d) directionality, (e) velocity, and (f) accumulated distance. Scatter plots represent individual experiments and the median for each group is represented as a horizontal bar. While individual or pooled cell samples varied in their migratory capacities between experiments, the median values were not significantly different. This is also exemplified by mean + SEM values for forward migration index (pooled 0.155 ± 0.030, range 0.04–0.30 and non-pooled 0.16 ± 0.02, range 0.10–0.27; p = 0.9 n = 8 and 7 respectively) and cell velocity (pooled 1.07 ± 0.04, range 0.92–1.29 μm/min and non-pooled 1.06 ± 0.11, range 0.77–0.11 μm/min; p = 0.5) (Mann Whitney test).

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: Comparison of chemotactic and chemokinetic responses of pooled versus non-pooled human cord blood CD133 + cells. Human CD133 + cells were isolated from umbilical cord blood and cultured as individual units (non-pooled; n = 7) or as pooled units (pooled; n = 8) in StemSpan medium containing SCF, Flt-3 ligand, IL-6 and TPO for 24 h before encapsulation into 1 mg/ml collagen I gel and seeding into the central chamber of a 3D chemotaxis μ-slide. Cell migration was tracked using timelapse microscopy and Image J software over 22 h in the presence of 1 μg/ml CXCL12. The chemotaxis and migration tool plug in was used to quantify (a) the percentage of cells migrating, (b) the displacement of the center of mass, (c) forward migration index, (d) directionality, (e) velocity, and (f) accumulated distance. Scatter plots represent individual experiments and the median for each group is represented as a horizontal bar. While individual or pooled cell samples varied in their migratory capacities between experiments, the median values were not significantly different. This is also exemplified by mean + SEM values for forward migration index (pooled 0.155 ± 0.030, range 0.04–0.30 and non-pooled 0.16 ± 0.02, range 0.10–0.27; p = 0.9 n = 8 and 7 respectively) and cell velocity (pooled 1.07 ± 0.04, range 0.92–1.29 μm/min and non-pooled 1.06 ± 0.11, range 0.77–0.11 μm/min; p = 0.5) (Mann Whitney test).

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Isolation, Cell Culture, Chemotaxis Assay, Migration, Microscopy, Software, MANN-WHITNEY

    CD133 + cell chemotaxis and chemokinesis as a function of time. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient as illustrated in Fig. 1 . Cell migration was imaged for 22 h by timelapse microscopy and tracked and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in after 4, 10, 18 and 22 h. (A) shows representative trajectory plots (a) 4 h, (b) 10 h, (c) 18 h and (d) 22 h (the blue cross shows the displacement of the center of mass) of cells exposed to 1 μg/ml CXCL12. The red and black lines indicate whether the cells finished their migration path below (towards 1 μg/ml CXCL12) or above their starting point on the x axis. The Rayleigh test indicate that the distribution of the cell end points was significantly inhomogeneous at all time points (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (p = 4.0 × 10 − 3 , p = 2.9 × 10 − 5 , p = 6.7 × 10 − 7 and 2.0 × 10 − 8 for (a)–(d) respectively. (B) shows the quantitative data for (a,b) accumulated distance, (c,d) velocity, (e,f) displacement of the center of mass, and (g,h) forward migration index, of cells exposed to 0 μg/ml CXCL12 (a,c,e,g) or 1 μg/ml CXCL12 (b,d,f,h) respectively. Values are means ± SEM for n = 3 independent experiments.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: CD133 + cell chemotaxis and chemokinesis as a function of time. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient as illustrated in Fig. 1 . Cell migration was imaged for 22 h by timelapse microscopy and tracked and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in after 4, 10, 18 and 22 h. (A) shows representative trajectory plots (a) 4 h, (b) 10 h, (c) 18 h and (d) 22 h (the blue cross shows the displacement of the center of mass) of cells exposed to 1 μg/ml CXCL12. The red and black lines indicate whether the cells finished their migration path below (towards 1 μg/ml CXCL12) or above their starting point on the x axis. The Rayleigh test indicate that the distribution of the cell end points was significantly inhomogeneous at all time points (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (p = 4.0 × 10 − 3 , p = 2.9 × 10 − 5 , p = 6.7 × 10 − 7 and 2.0 × 10 − 8 for (a)–(d) respectively. (B) shows the quantitative data for (a,b) accumulated distance, (c,d) velocity, (e,f) displacement of the center of mass, and (g,h) forward migration index, of cells exposed to 0 μg/ml CXCL12 (a,c,e,g) or 1 μg/ml CXCL12 (b,d,f,h) respectively. Values are means ± SEM for n = 3 independent experiments.

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Chemotaxis Assay, Cell Culture, Migration, Microscopy, Software

    The 3D μ-slide chemotaxis assay for human cord blood CD133 + hematopoietic stem/progenitor cells. (A) After 24 hour culture in StemSpan medium plus SCF, Flt-3 ligand, IL-6 and TPO, (a) human UCB CD133 + cells in a collagen I gel solution (1 mg/ml) were seeded into the central 3D μ-slide chamber prior to (b) the addition of StemSpan serum free medium alone (C0) or with a 1 μg/ml CXCL12 (C100) gradient plus or minus AMD3100 and cells captured by timelapse microscopy at 3–5 minute intervals. (B) Trajectory plots illustrate migrated cells at 22 h minus (a) or plus a CXCL12 gradient (b) or with CXCL12 in the presence of 10 μM AMD3100 (c). The red and black lines indicate whether the cells finished their migration path below or above their starting point on the x axis. Rayleigh test p values of p = 0.17, p = 5.7 × 10 − 8 and p = 0.054 for (a), (b) and (c) respectively indicate that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 alone (b). (C) Diagrammatic representation of a trajectory plot demonstrating methods for quantitating chemotactic and chemokinetic parameters. The diagrams in Figs. 1A and C were captured from the movie describing the assay system (MV_25_chemotaxis.flv) on the Ibidi website ( http://ibidi.com/support/movies/mv25/ ).

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: The 3D μ-slide chemotaxis assay for human cord blood CD133 + hematopoietic stem/progenitor cells. (A) After 24 hour culture in StemSpan medium plus SCF, Flt-3 ligand, IL-6 and TPO, (a) human UCB CD133 + cells in a collagen I gel solution (1 mg/ml) were seeded into the central 3D μ-slide chamber prior to (b) the addition of StemSpan serum free medium alone (C0) or with a 1 μg/ml CXCL12 (C100) gradient plus or minus AMD3100 and cells captured by timelapse microscopy at 3–5 minute intervals. (B) Trajectory plots illustrate migrated cells at 22 h minus (a) or plus a CXCL12 gradient (b) or with CXCL12 in the presence of 10 μM AMD3100 (c). The red and black lines indicate whether the cells finished their migration path below or above their starting point on the x axis. Rayleigh test p values of p = 0.17, p = 5.7 × 10 − 8 and p = 0.054 for (a), (b) and (c) respectively indicate that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 alone (b). (C) Diagrammatic representation of a trajectory plot demonstrating methods for quantitating chemotactic and chemokinetic parameters. The diagrams in Figs. 1A and C were captured from the movie describing the assay system (MV_25_chemotaxis.flv) on the Ibidi website ( http://ibidi.com/support/movies/mv25/ ).

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Chemotaxis Assay, Microscopy, Migration

    CXCL12 induces human cord blood CD133 + hematopoietic stem/progenitor cell chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient. (A) The results of 8 independent experiments are displayed as median values (horizontal bar) for individual experiments (scatter plots). Cell migration was tracked using Image J manual tracking software for 22 h and chemotactic and chemokinetic parameters quantified with the chemotaxis and migration tool plug-in for (a) the Rayleigh p value, (b) displacement of the center of mass (blue cross in Figs. 1 B and C), (c) forward migration index, (d) directionality, (e) velocity and (f) accumulated distance. Interestingly, there were significant differences in chemotaxis between the chambers containing medium alone and those containing CXCL12, with the following respective changes (mean ± SEM) in Rayleigh values (0.336 ± 0.073 vs. 0.001 ± 0.001; p = 0.0002), displacement of center of mass (10.82 ± 11.25 vs. 189.00 ± 44.26; p = 0.0002) and forward migration index (0.015 ± 0.008 vs. 0.126 ± 0.024; p = 0.0004) over 8 independent experiments, but no significant difference in cell velocity or accumulated distance without or with a CXCL12 gradient (p = 0.7128 and p = 0.2345 respectively). Statistics for chemotactic and chemokinetic data were calculated using the Mann–Whitney test with *p

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: CXCL12 induces human cord blood CD133 + hematopoietic stem/progenitor cell chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 0 μg/ml or 1 μg/ml CXCL12 to form a chemokine gradient. (A) The results of 8 independent experiments are displayed as median values (horizontal bar) for individual experiments (scatter plots). Cell migration was tracked using Image J manual tracking software for 22 h and chemotactic and chemokinetic parameters quantified with the chemotaxis and migration tool plug-in for (a) the Rayleigh p value, (b) displacement of the center of mass (blue cross in Figs. 1 B and C), (c) forward migration index, (d) directionality, (e) velocity and (f) accumulated distance. Interestingly, there were significant differences in chemotaxis between the chambers containing medium alone and those containing CXCL12, with the following respective changes (mean ± SEM) in Rayleigh values (0.336 ± 0.073 vs. 0.001 ± 0.001; p = 0.0002), displacement of center of mass (10.82 ± 11.25 vs. 189.00 ± 44.26; p = 0.0002) and forward migration index (0.015 ± 0.008 vs. 0.126 ± 0.024; p = 0.0004) over 8 independent experiments, but no significant difference in cell velocity or accumulated distance without or with a CXCL12 gradient (p = 0.7128 and p = 0.2345 respectively). Statistics for chemotactic and chemokinetic data were calculated using the Mann–Whitney test with *p

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Chemotaxis Assay, Cell Culture, Migration, Software, MANN-WHITNEY

    AMD3100 inhibits chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 1 μg/ml CXCL12 to form a chemokine gradient with or without AMD3100. Histograms Illustrate the effect of adding 0, 1 μM or 10 μM AMD3100 with 1 μg/ml CXCL12 for the chemotactic or chemokinetic responses of forward migration index (A) and displacement of the center of mass (B) or velocity (C) and accumulated distance (D). Controls contained media alone. Values are means ± SEM for n ≥ 3 independent experiments. Notably, with the addition of AMD3100, there was a significant decrease in the Rayleigh test p values (E), in displacement of the center of mass (mean ± SEM values of 126.5 μm ± 21.1 for no AMD3100 to 24.7 μm ± 18.1 for 1 μM AMD3100 and 7.5 μm ± 10.6 for 10 μM AMD3100) and in forward migration index (mean ± SEM values of 0.11 ± 0.02 with no AMD3100 and 0.01 ± 0.01 for 1 μM AMD3100 and 0.01 ± 0.01 for 10 μM AMD3100) towards CXCL12 compared to absent AMD3100 (p

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: AMD3100 inhibits chemotaxis but not chemokinesis. Human UCB CD133 + cells were cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h, encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides, prior to adding 1 μg/ml CXCL12 to form a chemokine gradient with or without AMD3100. Histograms Illustrate the effect of adding 0, 1 μM or 10 μM AMD3100 with 1 μg/ml CXCL12 for the chemotactic or chemokinetic responses of forward migration index (A) and displacement of the center of mass (B) or velocity (C) and accumulated distance (D). Controls contained media alone. Values are means ± SEM for n ≥ 3 independent experiments. Notably, with the addition of AMD3100, there was a significant decrease in the Rayleigh test p values (E), in displacement of the center of mass (mean ± SEM values of 126.5 μm ± 21.1 for no AMD3100 to 24.7 μm ± 18.1 for 1 μM AMD3100 and 7.5 μm ± 10.6 for 10 μM AMD3100) and in forward migration index (mean ± SEM values of 0.11 ± 0.02 with no AMD3100 and 0.01 ± 0.01 for 1 μM AMD3100 and 0.01 ± 0.01 for 10 μM AMD3100) towards CXCL12 compared to absent AMD3100 (p

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Chemotaxis Assay, Cell Culture, Migration

    The CD133 + cell chemotactic response to increasing concentrations of CXCL12. Human UCB CD133 + cells cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h were encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides. 0 μg/ml, 0.2 μg/ml, 1 μg/ml or 2 μg/ml CXCL12 were added to create a chemokine gradient. The cells were imaged over 22 h by timelapse microscopy and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in. (A) Representative trajectory plots after CD133 + cells were exposed to differing concentrations of CXCL12. The red and black lines indicate whether the cells finished their migration path below or above (towards differing CXCL12 concentrations) their starting point on the x axis. Rayleigh test p values were p = 0.16, p = 6.1 × 10 − 5 , p = 4.0 × 10 − 10 and p = 1.0 × 10 − 7 respectively for figures (a)–(d) indicating that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (b)–(d) and that this was most significant with 1 μg/ml (c). (B) shows the quantitative data for (a) displacement of center of mass (257.8 μm ± 112.9 for 1 μg/ml compared to no CXCL12 of 48.8 μm ± 8.9), (b) forward migration index (0.18 ± 0.05 for 1 μg/ml compared to no CXCL12 of 0.02 ± 0.00), (c) cell velocity and (d) accumulated distance as a function of CXCL12 concentration. Values are means ± SEM for n = 3 independent experiments.

    Journal: Stem Cell Research

    Article Title: A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133+ stem/progenitor cells

    doi: 10.1016/j.scr.2013.04.006

    Figure Lengend Snippet: The CD133 + cell chemotactic response to increasing concentrations of CXCL12. Human UCB CD133 + cells cultured in StemSpan medium with SCF, Flt-3 ligand, IL-6 and TPO for 24 h were encapsulated in 1 mg/ml collagen I gel and seeded into the central chamber of the 3D chemotaxis μ-slides. 0 μg/ml, 0.2 μg/ml, 1 μg/ml or 2 μg/ml CXCL12 were added to create a chemokine gradient. The cells were imaged over 22 h by timelapse microscopy and analyzed using Image J manual tracking software and the chemotaxis and migration tool plug-in. (A) Representative trajectory plots after CD133 + cells were exposed to differing concentrations of CXCL12. The red and black lines indicate whether the cells finished their migration path below or above (towards differing CXCL12 concentrations) their starting point on the x axis. Rayleigh test p values were p = 0.16, p = 6.1 × 10 − 5 , p = 4.0 × 10 − 10 and p = 1.0 × 10 − 7 respectively for figures (a)–(d) indicating that the distribution of the cell end points was only significantly inhomogeneous (i.e. distributed towards the chemoattractant) in the presence of CXCL12 (b)–(d) and that this was most significant with 1 μg/ml (c). (B) shows the quantitative data for (a) displacement of center of mass (257.8 μm ± 112.9 for 1 μg/ml compared to no CXCL12 of 48.8 μm ± 8.9), (b) forward migration index (0.18 ± 0.05 for 1 μg/ml compared to no CXCL12 of 0.02 ± 0.00), (c) cell velocity and (d) accumulated distance as a function of CXCL12 concentration. Values are means ± SEM for n = 3 independent experiments.

    Article Snippet: The reservoirs either side of the observation chamber were then filled slowly using a beveled pipette tip with up to 70 μl serum free StemSpan media (control media; Stem Cell Technologies) or chemo-attractant media comprising StemSpan media plus CXCL12 (Peprotech, tebu-bio, Peterborough, England) in order to establish a CXCL12 gradient over the observation area ( A(b)).

    Techniques: Cell Culture, Chemotaxis Assay, Microscopy, Software, Migration, Concentration Assay

    IgG1 IgLON5 antibodies internalize IgLON5 clusters. Hippocampal neurons treated for 3 days with total IgG, IgG1, and IgG4 IgLON5 antibodies. The immunofluorescence strategy to differentiate surface and internal human IgG bound to IgLON5 is conducted as in Fig. 8 . The IgG1 antibodies alone could reproduce the same effects seen with the total IgG; meanwhile, the IgG4 did not produce internalization. Scale bar = 5 μm

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: IgG1 IgLON5 antibodies internalize IgLON5 clusters. Hippocampal neurons treated for 3 days with total IgG, IgG1, and IgG4 IgLON5 antibodies. The immunofluorescence strategy to differentiate surface and internal human IgG bound to IgLON5 is conducted as in Fig. 8 . The IgG1 antibodies alone could reproduce the same effects seen with the total IgG; meanwhile, the IgG4 did not produce internalization. Scale bar = 5 μm

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Immunofluorescence

    IgLON5 antibodies irreversible decrease IgLON5 clusters on cell surface. a Immunofluorescence on hippocampal neurons treated for 7 days (14DIV to 21DIV) with control or patient with anti-IgLON5 IgG. The surface clusters are drastically reduced by the anti-IgLON5 IgG, and the effect is irreversible because 7 days after removing the anti-IgLON5 IgG the reduced number of surface IgLON5 clusters persisted. Scale bar = 5 μm. b Quantification of the decrease of IgLON5 clusters on the dendrite surface in a time-course treatment of 1, 3, and 7 days, and 7 days after removing the anti-IgLON5 IgG. *** p

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: IgLON5 antibodies irreversible decrease IgLON5 clusters on cell surface. a Immunofluorescence on hippocampal neurons treated for 7 days (14DIV to 21DIV) with control or patient with anti-IgLON5 IgG. The surface clusters are drastically reduced by the anti-IgLON5 IgG, and the effect is irreversible because 7 days after removing the anti-IgLON5 IgG the reduced number of surface IgLON5 clusters persisted. Scale bar = 5 μm. b Quantification of the decrease of IgLON5 clusters on the dendrite surface in a time-course treatment of 1, 3, and 7 days, and 7 days after removing the anti-IgLON5 IgG. *** p

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Immunofluorescence

    IgLON5 antibodies produce internalization of IgLON5 clusters. a Panel 1: Hippocampal neurons treated for 7 days with IgG-positive IgLON5 antibodies. Panel 2: IgG bound to IgLON5 on the dendrite surface labeled live with an excess of anti-human IgG Alexa Fluor 594 ( red ). Panel 3A: The saturation of the neuronal surface prevents that the anti-human IgG Alexa Fluor 488 ( green ) attaches to the surface. Panel 3B: After the neuron is permeabilized and incubated with the anti-human IgG Alexa Fluor 488, the green fluorescence localizes the human IgG attached to internalized IgLON5 clusters. Scale bar = 5 μm. b The internalization of IgLON5 clusters is a time-dependent effect and parallels a decrease of IgLON5 clusters on the surface of the dendrite. c Quantification of the number of IgLON5 clusters remaining on the cell surface and internalized after the treatment

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: IgLON5 antibodies produce internalization of IgLON5 clusters. a Panel 1: Hippocampal neurons treated for 7 days with IgG-positive IgLON5 antibodies. Panel 2: IgG bound to IgLON5 on the dendrite surface labeled live with an excess of anti-human IgG Alexa Fluor 594 ( red ). Panel 3A: The saturation of the neuronal surface prevents that the anti-human IgG Alexa Fluor 488 ( green ) attaches to the surface. Panel 3B: After the neuron is permeabilized and incubated with the anti-human IgG Alexa Fluor 488, the green fluorescence localizes the human IgG attached to internalized IgLON5 clusters. Scale bar = 5 μm. b The internalization of IgLON5 clusters is a time-dependent effect and parallels a decrease of IgLON5 clusters on the surface of the dendrite. c Quantification of the number of IgLON5 clusters remaining on the cell surface and internalized after the treatment

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Labeling, Incubation, Fluorescence

    Comparison of the time-dependent decrease of neuronal cell surface IgLON5 clusters after treatment with total IgG of two patients with different levels of anti-IgLON5 IgG1 antibodies. On days 1 and 3, treatment with IgG of the patient with the lower level of IgG1 IgLON5 antibodies (7 % of total IgLON5 antibodies) produced a similar decrease of IgLON5 clusters as the IgG of the patient with average level of IgG1 antibodies (23 % of total IgLON5 antibodies). (** p = 0.001, **** p

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: Comparison of the time-dependent decrease of neuronal cell surface IgLON5 clusters after treatment with total IgG of two patients with different levels of anti-IgLON5 IgG1 antibodies. On days 1 and 3, treatment with IgG of the patient with the lower level of IgG1 IgLON5 antibodies (7 % of total IgLON5 antibodies) produced a similar decrease of IgLON5 clusters as the IgG of the patient with average level of IgG1 antibodies (23 % of total IgLON5 antibodies). (** p = 0.001, **** p

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Produced

    IgLON5 IgG1, but not IgG4 antibodies, cause a reduction of surface IgLON5 clusters. a Hippocampal neurons were treated for 3 days with IgG1 or IgG4 fractions from control serum or from a patient with IgLON5 antibodies and immunostained for surface antibody-bound IgLON5. Scale bar = 5 μm. b Quantification of IgLON5 clusters. IgLON5 clusters were significantly reduced after treatment with patient IgG1 compared with control IgG1 or patient and control IgG4. * p

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: IgLON5 IgG1, but not IgG4 antibodies, cause a reduction of surface IgLON5 clusters. a Hippocampal neurons were treated for 3 days with IgG1 or IgG4 fractions from control serum or from a patient with IgLON5 antibodies and immunostained for surface antibody-bound IgLON5. Scale bar = 5 μm. b Quantification of IgLON5 clusters. IgLON5 clusters were significantly reduced after treatment with patient IgG1 compared with control IgG1 or patient and control IgG4. * p

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques:

    Epitope analysis of IgLON5 antibodies. The diagrams depict the complete IgLON5 ( bottom ) and mutated clones ( left ). Immunofluorescence of transfected HEK293 cells with the full length or the mutant IgLON5 clones ( right ). Rows correspond to the indicated clone in the diagram. The serum with IgLON5 antibodies ( green ) only targets the clones containing the immunoglobulin-like domain 2 (Ig2, left column ). IgLON5 commercial antibody immunoreactivity is shown in the central column (in red ) and both reactivities are shown merged in the right column . Nuclei counterstained with DAPI ( blue ). Scale bar = 10 μm. The (*) indicates the epitope recognized by the IgLON5 commercial antibody

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: Epitope analysis of IgLON5 antibodies. The diagrams depict the complete IgLON5 ( bottom ) and mutated clones ( left ). Immunofluorescence of transfected HEK293 cells with the full length or the mutant IgLON5 clones ( right ). Rows correspond to the indicated clone in the diagram. The serum with IgLON5 antibodies ( green ) only targets the clones containing the immunoglobulin-like domain 2 (Ig2, left column ). IgLON5 commercial antibody immunoreactivity is shown in the central column (in red ) and both reactivities are shown merged in the right column . Nuclei counterstained with DAPI ( blue ). Scale bar = 10 μm. The (*) indicates the epitope recognized by the IgLON5 commercial antibody

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Clone Assay, Immunofluorescence, Transfection, Mutagenesis

    Deglycosylation assay. Western blot of human cerebellum protein extract treated with N-Gly (N-glycosidase), O-Gly (O-glycosidase), and/or NA (neuraminidase) and probed with a commercial rabbit anti-human IgLON5 antibody. The IgLON5 protein is completely deglycosylated only when the N-linked glycans are removed reaching its predicted molecular weight of 36 kDa according to the amino acid sequence

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: Deglycosylation assay. Western blot of human cerebellum protein extract treated with N-Gly (N-glycosidase), O-Gly (O-glycosidase), and/or NA (neuraminidase) and probed with a commercial rabbit anti-human IgLON5 antibody. The IgLON5 protein is completely deglycosylated only when the N-linked glycans are removed reaching its predicted molecular weight of 36 kDa according to the amino acid sequence

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Western Blot, Molecular Weight, Sequencing

    Analysis of IgLON5 antibody subclasses. a Example of anti-IgLON5 IgG subclasses in three different sera. HEK293 cells were transfected with IgLON5 and incubated with three sera with IgLON5 antibodies followed by antibodies against total IgG or specific IgG subclasses. Positive immunoreactivity is shown in green . Nuclei counterstained with DAPI ( blue ). Scale bar = 10 μm. b IgG subclass percentages of IgLON5 antibodies in the 15 positive serum samples analyzed by flow cytometry

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: Analysis of IgLON5 antibody subclasses. a Example of anti-IgLON5 IgG subclasses in three different sera. HEK293 cells were transfected with IgLON5 and incubated with three sera with IgLON5 antibodies followed by antibodies against total IgG or specific IgG subclasses. Positive immunoreactivity is shown in green . Nuclei counterstained with DAPI ( blue ). Scale bar = 10 μm. b IgG subclass percentages of IgLON5 antibodies in the 15 positive serum samples analyzed by flow cytometry

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Transfection, Incubation, Flow Cytometry, Cytometry

    Deglycosylated IgLON5 is recognized by human IgLON5 antibodies. A Anti-IgLON5 immunoreactivity in rat brain sections is totally abrogated when the anti-IgLON5-positive serum is preabsorbed with glycosylated ( a ) or deglycosylated protein extracts from transfected HEK293 with IgLON5 ( c ). The immunoreactivity of IgLON5 antibodies is present when the serum is preabsorbed with an unrelated antigen glycosylated ( b ) or deglycosylated ( d ). Scale bar = 1000 μm. B a Immunofluorescence of HEK293 cells transfected with IgLON5. Prevention of N-glycosylation with tunicamycin treatment does not affect the reactivity of patients’ antibodies with IgLON5. Scale bar = 10 μm. b Western blot of extracts of the indicated IgLON5-HEK293 cells shown in ( a ), demonstrating a shift of the molecular weight (from ~52 to 36 kDa) consistent with the deglycosylation of the protein. The findings confirm that the reactivity of patients’ serum antibodies with tunicamycin-treated cells of A was directed against deglycosylated epitopes

    Journal: Journal of Neuroinflammation

    Article Title: Cellular investigations with human antibodies associated with the anti-IgLON5 syndrome

    doi: 10.1186/s12974-016-0689-1

    Figure Lengend Snippet: Deglycosylated IgLON5 is recognized by human IgLON5 antibodies. A Anti-IgLON5 immunoreactivity in rat brain sections is totally abrogated when the anti-IgLON5-positive serum is preabsorbed with glycosylated ( a ) or deglycosylated protein extracts from transfected HEK293 with IgLON5 ( c ). The immunoreactivity of IgLON5 antibodies is present when the serum is preabsorbed with an unrelated antigen glycosylated ( b ) or deglycosylated ( d ). Scale bar = 1000 μm. B a Immunofluorescence of HEK293 cells transfected with IgLON5. Prevention of N-glycosylation with tunicamycin treatment does not affect the reactivity of patients’ antibodies with IgLON5. Scale bar = 10 μm. b Western blot of extracts of the indicated IgLON5-HEK293 cells shown in ( a ), demonstrating a shift of the molecular weight (from ~52 to 36 kDa) consistent with the deglycosylation of the protein. The findings confirm that the reactivity of patients’ serum antibodies with tunicamycin-treated cells of A was directed against deglycosylated epitopes

    Article Snippet: To determine the IgG subclasses by flow cytometry, IgLON5 transfected or non-transfected HEK293 cells were mildly trypsinized and incubated with a mix of IgLON5-positive human serum samples (1/50) and the rabbit-anti-human IgLON5 commercial antibody (Abcam) (1/1000) for 20 min at 4 °C in DMEM (Dulbecco’s modified Eagle medium, Thermo Fisher Scientific) plus 1 % bovine serum albumin (BSA).

    Techniques: Transfection, Immunofluorescence, Western Blot, Molecular Weight

    IE2 is degraded in the absence of its E3 ligase activity. Wild type (WT) IE2 and IE2C230S (containing a RING domain mutation)-expressing recombinant viruses were transduced into Vero E6 cells together with KNK437 treatment at various concentrations. The cell extracts were collected at 48 hpt for the detection of IE2 protein levels.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: IE2 is degraded in the absence of its E3 ligase activity. Wild type (WT) IE2 and IE2C230S (containing a RING domain mutation)-expressing recombinant viruses were transduced into Vero E6 cells together with KNK437 treatment at various concentrations. The cell extracts were collected at 48 hpt for the detection of IE2 protein levels.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Activity Assay, Mutagenesis, Expressing, Recombinant

    IE2 associates with HSPs. vAcIE2-transduced Vero E6 cells were fixed and examined by immunofluorescence staining at 48 hpt. Nuclei have been enlarged to more clearly present the interaction between IE2, HSP70 and HSP90. (A) Panels a to d show that HSP70 formed dots and was scattered within the nucleus. Larger foci of HSP70 closely associated with IE2 nuclear bodies. Panels e to h show that HSP90 also formed foci and was closely associated with IE2 nuclear bodies. (B) The IE2 protein complex was immunoprecipitated using an anti-histidine antibody from nuclear extracts of the vAcIE2-transduced Vero E6 cells. Nuclear extracts of the vAcE-transduced Vero E6 cells were used as a control. HSP70, actin and IE2 were detected by Western blotting using the corresponding antibody. FT: flow-through, W: wash, E: eluted fraction.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: IE2 associates with HSPs. vAcIE2-transduced Vero E6 cells were fixed and examined by immunofluorescence staining at 48 hpt. Nuclei have been enlarged to more clearly present the interaction between IE2, HSP70 and HSP90. (A) Panels a to d show that HSP70 formed dots and was scattered within the nucleus. Larger foci of HSP70 closely associated with IE2 nuclear bodies. Panels e to h show that HSP90 also formed foci and was closely associated with IE2 nuclear bodies. (B) The IE2 protein complex was immunoprecipitated using an anti-histidine antibody from nuclear extracts of the vAcIE2-transduced Vero E6 cells. Nuclear extracts of the vAcE-transduced Vero E6 cells were used as a control. HSP70, actin and IE2 were detected by Western blotting using the corresponding antibody. FT: flow-through, W: wash, E: eluted fraction.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Flow Cytometry

    IE2 stimulates HSC70 expression during baculovirus infection in insect cells. (A) Total RNA was collected from vAcE-infected Sf21 cells at 0, 2, 4, 6, 8 hpi, and the expression patterns of ie2 (A1) and hsc70 (A2) genes were quantified by RT-qPCR using specific primers. (B) Plasmids expressing the ie2 and egfp genes were transfected into Sf21 cells in the absence of virus. An up-regulated level of hsc70 transcripts can be observed using RT-qPCR (B1). IE2 nuclear bodies can also be detected within the nucleus of the transfected cells (B2). In A1, A2 and B1, the relative ie2 and hsc70 levels were individually normalized to actin gene levels.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: IE2 stimulates HSC70 expression during baculovirus infection in insect cells. (A) Total RNA was collected from vAcE-infected Sf21 cells at 0, 2, 4, 6, 8 hpi, and the expression patterns of ie2 (A1) and hsc70 (A2) genes were quantified by RT-qPCR using specific primers. (B) Plasmids expressing the ie2 and egfp genes were transfected into Sf21 cells in the absence of virus. An up-regulated level of hsc70 transcripts can be observed using RT-qPCR (B1). IE2 nuclear bodies can also be detected within the nucleus of the transfected cells (B2). In A1, A2 and B1, the relative ie2 and hsc70 levels were individually normalized to actin gene levels.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Expressing, Infection, Quantitative RT-PCR, Transfection

    IE2 protein level is restored with the combination treatment of KNK437 and MG132. vAcIE2-transduced Vero E6 cells were treated with KNK437 (5 μM) and/or MG132 (2.5 μM) in various combinations. (A-B) Cell extracts were prepared at 48 hpt to detect the presence of IE2, ubiquitin and GAPDH proteins. Western blot analysis showed a significant recovery of IE2 protein levels in the presence of the proteasome inhibitor, MG132. ND: no drug, K: KNK437, M: MG132, KM: KNK437+MG132. (C) vAcIE2-transduced Vero E6 cells were fixed and examined by immunofluorescence staining. Panels a to d show that at 48 hpt, IE2 formed mature nuclear bodies that included a high concentration of G-actin (green) and enlarged, activated Pol II dots (red). KNK437 treatment alone (panels e to h ) led to a failure in IE2 nuclear body formation as well as G-actin and activated Pol II recruitment, whereas for KNK437 in combination with MG132 (panels i to l ), mature IE2 nuclear bodies could again be observed. (D) vAcIE2 and vAcL co-transduced Vero E6 cells were treated with drugs at the indicated combinations and collected at 48 hpt. The luciferase assay shows that IE2 trans -activation activity was also restored in the presence of MG132.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: IE2 protein level is restored with the combination treatment of KNK437 and MG132. vAcIE2-transduced Vero E6 cells were treated with KNK437 (5 μM) and/or MG132 (2.5 μM) in various combinations. (A-B) Cell extracts were prepared at 48 hpt to detect the presence of IE2, ubiquitin and GAPDH proteins. Western blot analysis showed a significant recovery of IE2 protein levels in the presence of the proteasome inhibitor, MG132. ND: no drug, K: KNK437, M: MG132, KM: KNK437+MG132. (C) vAcIE2-transduced Vero E6 cells were fixed and examined by immunofluorescence staining. Panels a to d show that at 48 hpt, IE2 formed mature nuclear bodies that included a high concentration of G-actin (green) and enlarged, activated Pol II dots (red). KNK437 treatment alone (panels e to h ) led to a failure in IE2 nuclear body formation as well as G-actin and activated Pol II recruitment, whereas for KNK437 in combination with MG132 (panels i to l ), mature IE2 nuclear bodies could again be observed. (D) vAcIE2 and vAcL co-transduced Vero E6 cells were treated with drugs at the indicated combinations and collected at 48 hpt. The luciferase assay shows that IE2 trans -activation activity was also restored in the presence of MG132.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Western Blot, Immunofluorescence, Staining, Concentration Assay, Luciferase, Activation Assay, Activity Assay

    HSPs stabilize the IE2 protein during baculovirus infection. vABiIE1WF-infected Sf21 cells were treated with DMSO or KNK437, and the samples were collected at the indicated times. (A) Western blot analysis showing the expression pattern of IE2 and GAPDH without (A1) and with (A2) KNK437 treatment. KNK437 treatment led to a significantly decreased level of IE2 at various time-points. (B) Immunofluorescence staining assays followed by MetaMorph analysis show that both the intensity and formation efficiency of the IE2 and IE1 nuclear bodies decreased without (B1) and with (B2) KNK437 treatment.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: HSPs stabilize the IE2 protein during baculovirus infection. vABiIE1WF-infected Sf21 cells were treated with DMSO or KNK437, and the samples were collected at the indicated times. (A) Western blot analysis showing the expression pattern of IE2 and GAPDH without (A1) and with (A2) KNK437 treatment. KNK437 treatment led to a significantly decreased level of IE2 at various time-points. (B) Immunofluorescence staining assays followed by MetaMorph analysis show that both the intensity and formation efficiency of the IE2 and IE1 nuclear bodies decreased without (B1) and with (B2) KNK437 treatment.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Infection, Western Blot, Expressing, Immunofluorescence, Staining

    Schematic diagram of IE2 and heat shock protein interactions during baculovirus infection. (A) Upon virus infection, IE2 is expressed and stimulates heat shock protein expression. The elevated levels of heat shock proteins further sustain IE2 transcription center formation, most likely by protecting it from degradation. The IE2 transcription centers activate or recruit several additional viral factors including IE1, Lef3 and DBP [ 20 ], allowing the virus to maximize its amplification in the infected cells. (B) In the presence of KNK437-the heat shock response inhibitor-IE2 is rapidly degraded through the ubiquitin-proteasome pathway and fails to form mature transcription centers. The lack of IE2 nuclear body formation eventually contributes to lower levels of virus titer.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: Schematic diagram of IE2 and heat shock protein interactions during baculovirus infection. (A) Upon virus infection, IE2 is expressed and stimulates heat shock protein expression. The elevated levels of heat shock proteins further sustain IE2 transcription center formation, most likely by protecting it from degradation. The IE2 transcription centers activate or recruit several additional viral factors including IE1, Lef3 and DBP [ 20 ], allowing the virus to maximize its amplification in the infected cells. (B) In the presence of KNK437-the heat shock response inhibitor-IE2 is rapidly degraded through the ubiquitin-proteasome pathway and fails to form mature transcription centers. The lack of IE2 nuclear body formation eventually contributes to lower levels of virus titer.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Infection, Expressing, Amplification

    HSPs positively regulate IE2 transaction activity. (A) vAcIE2 and vAcL co-transduced Vero E6 cells were treated with KNK437 at various concentrations, followed by luciferase assays at 48 hpt. The results show that IE2 trans -activation activity on the CMV promoter was down-regulated in a dose-dependent manner. KNK437 did not have a negative effect on the control group represented by vAcE transduction. (B) siRNA against hsp70 (B1) and hsp90 (B2) genes were transfected into Vero E6 cells, followed by vAcIE2 and vAcL co-transduction. A luciferase assay at 48 hpt detected negative effects on the IE2 trans -activation of the CMV promoter. (C) siRNA against hsp70 (C1), hsp90 (C2) and negative control siRNA against a scrambled sequence (Santa Cruz) were transfected into Vero E6 cells, followed by vAcIE2 or vAcE transduction. Cell extracts were harvested at 48 hpt and analyzed by Western blotting. (D) PGA 1 , which stimulates the heat shock response, further enhanced the IE2 trans -activation effect on the CMV promoter in a dose-dependent manner.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: HSPs positively regulate IE2 transaction activity. (A) vAcIE2 and vAcL co-transduced Vero E6 cells were treated with KNK437 at various concentrations, followed by luciferase assays at 48 hpt. The results show that IE2 trans -activation activity on the CMV promoter was down-regulated in a dose-dependent manner. KNK437 did not have a negative effect on the control group represented by vAcE transduction. (B) siRNA against hsp70 (B1) and hsp90 (B2) genes were transfected into Vero E6 cells, followed by vAcIE2 and vAcL co-transduction. A luciferase assay at 48 hpt detected negative effects on the IE2 trans -activation of the CMV promoter. (C) siRNA against hsp70 (C1), hsp90 (C2) and negative control siRNA against a scrambled sequence (Santa Cruz) were transfected into Vero E6 cells, followed by vAcIE2 or vAcE transduction. Cell extracts were harvested at 48 hpt and analyzed by Western blotting. (D) PGA 1 , which stimulates the heat shock response, further enhanced the IE2 trans -activation effect on the CMV promoter in a dose-dependent manner.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Activity Assay, Luciferase, Activation Assay, Transduction, Transfection, Negative Control, Sequencing, Western Blot

    Purification of the IE2 nuclear bodies. Fractions containing IE2 protein were spotted onto poly-L-lysine-coated glass slides and immobilized with antibody and DNaseI for the detection of IE2 and G-actin, respectively. Panels a to d show that numerous IE2 nuclear bodies maintained their integrity after the purification procedure. Panels e to h show that the fraction largely contains IE2 nuclear bodies with little other visible cell debris.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: Purification of the IE2 nuclear bodies. Fractions containing IE2 protein were spotted onto poly-L-lysine-coated glass slides and immobilized with antibody and DNaseI for the detection of IE2 and G-actin, respectively. Panels a to d show that numerous IE2 nuclear bodies maintained their integrity after the purification procedure. Panels e to h show that the fraction largely contains IE2 nuclear bodies with little other visible cell debris.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Purification

    IE2 stimulates HSP70 and HSP90 expression in Vero E6 cells. Total RNA was extracted from vAcIE2-transduced Vero E6 cells at 6 hpt, and the transcripts of hsp70 (A) and hsp90 (B) were quantified by real-time quantitative PCR. The expression levels of hsp70 and hsp90 have been normalized to that of 18s rRNA. Total RNA from vAcE-transduced and non-transduced cells served as the controls.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: IE2 stimulates HSP70 and HSP90 expression in Vero E6 cells. Total RNA was extracted from vAcIE2-transduced Vero E6 cells at 6 hpt, and the transcripts of hsp70 (A) and hsp90 (B) were quantified by real-time quantitative PCR. The expression levels of hsp70 and hsp90 have been normalized to that of 18s rRNA. Total RNA from vAcE-transduced and non-transduced cells served as the controls.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    HSPs stabilize the IE2 protein. (A) Vero E6 cells were transduced with vAcIE2 virus together with KNK437 treatment at various concentrations. The transduced cells were fixed at 48 hpt for the detection of IE2 nuclear body formation. Immunofluorescence staining assays showed that, in the presence of KNK437 at concentrations up to 5 μM, smaller and fewer IE2 nuclear bodies were observed. Smaller IE2 nuclear bodies also failed to incorporate G-actin into the mature nuclear body structure. (B) vAcIE2-transduced cells were collected at 48 hpt for the detection of IE2 protein. Western blot analysis showed that the higher the concentration of KNK437, corresponding to greater inhibition of the heat shock response, the lower the levels of IE2 protein.

    Journal: PLoS ONE

    Article Title: Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning

    doi: 10.1371/journal.pone.0148578

    Figure Lengend Snippet: HSPs stabilize the IE2 protein. (A) Vero E6 cells were transduced with vAcIE2 virus together with KNK437 treatment at various concentrations. The transduced cells were fixed at 48 hpt for the detection of IE2 nuclear body formation. Immunofluorescence staining assays showed that, in the presence of KNK437 at concentrations up to 5 μM, smaller and fewer IE2 nuclear bodies were observed. Smaller IE2 nuclear bodies also failed to incorporate G-actin into the mature nuclear body structure. (B) vAcIE2-transduced cells were collected at 48 hpt for the detection of IE2 protein. Western blot analysis showed that the higher the concentration of KNK437, corresponding to greater inhibition of the heat shock response, the lower the levels of IE2 protein.

    Article Snippet: For Western blotting, anti-IE2 serum (1:3000), anti-HSP70 (1:3000, Genetex GTX111088), anti-Ubiquitin (1:1000, Santa Cruz sc-8017), anti-actin antibody (1:3000, Genetex GTX109639), or anti-GAPDH (1:3000, Genetex GTX100118) were used for the detection of target protein.

    Techniques: Transduction, Immunofluorescence, Staining, Western Blot, Concentration Assay, Inhibition

    The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human keratinocytes

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: The src kinase inhibitor and dominant negative src and fyn block calcium-stimulated PI3K-p85α phosphorylation and PI3K activity, whereas PI3K inhibition does not affect calcium stimulation of either src or fyn activity. Cultured human keratinocytes

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Dominant Negative Mutation, Blocking Assay, Activity Assay, Inhibition, Cell Culture

    PI3K inhibition blocks the stimulation of PLC-γ1 activity by calcium. Cultured human keratinocytes were preincubated with 10 μM LY294002 (+) or vehicle DMSO (–) for 1 h. Cells were then stimulated with 1.2 mM calcium for 1 h, and

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: PI3K inhibition blocks the stimulation of PLC-γ1 activity by calcium. Cultured human keratinocytes were preincubated with 10 μM LY294002 (+) or vehicle DMSO (–) for 1 h. Cells were then stimulated with 1.2 mM calcium for 1 h, and

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Inhibition, Planar Chromatography, Activity Assay, Cell Culture

    The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn block calcium-induced involucrin and transglutaminase expression. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2)

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn block calcium-induced involucrin and transglutaminase expression. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2)

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Dominant Negative Mutation, Blocking Assay, Expressing, Cell Culture

    Calcium stimulates PI3K-p85α phosphorylation, PI3K activity, and Akt phosphorylation. (A). Cultured human keratinocytes were treated with 1.2 mM calcium for the indicated times. The protein levels of PI3K-p85α and its phosphorylated form

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium stimulates PI3K-p85α phosphorylation, PI3K activity, and Akt phosphorylation. (A). Cultured human keratinocytes were treated with 1.2 mM calcium for the indicated times. The protein levels of PI3K-p85α and its phosphorylated form

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Activity Assay, Cell Culture

    Calcium does not stimulate the phosphorylation of PLC-γ1. Cultured human keratinocytes were treated with 1.2 mM calcium or 50 ng/ml EGF. The cells were harvested at the time points indicated. The protein levels of PLC-γ1 and its phosphorylated

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium does not stimulate the phosphorylation of PLC-γ1. Cultured human keratinocytes were treated with 1.2 mM calcium or 50 ng/ml EGF. The cells were harvested at the time points indicated. The protein levels of PLC-γ1 and its phosphorylated

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Cell Culture

    Calcium stimulates PLC-γ1 activity. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated after the addition of calcium. The activity of PLC-γ1 was assayed as described in

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium stimulates PLC-γ1 activity. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated after the addition of calcium. The activity of PLC-γ1 was assayed as described in

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Activity Assay, Cell Culture

    The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn reduce calcium-stimulated PLC-γ1 activity. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2) or its inactive

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: The src family kinase inhibitor PP2 and the combination of dominant negative src and fyn reduce calcium-stimulated PLC-γ1 activity. (A) Cultured human keratinocytes were treated with a specific src family kinase inhibitor (PP2) or its inactive

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Dominant Negative Mutation, Planar Chromatography, Activity Assay, Cell Culture

    Calcium stimulates src and fyn activities. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated. The activities of src (A) and fyn (B) were assayed as described in Materials and Methods and

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Calcium stimulates src and fyn activities. Cultured human keratinocytes were treated with 1.2 mM calcium. The cells were harvested at the time points indicated. The activities of src (A) and fyn (B) were assayed as described in Materials and Methods and

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Cell Culture

    Proposed model for the signaling pathway of PLC-γ1 activation mediating calcium-induced human keratinocyte differentiation. Calcium (Ca 2+ ) stimulates src and fyn , which activate PI3K via

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: Proposed model for the signaling pathway of PLC-γ1 activation mediating calcium-induced human keratinocyte differentiation. Calcium (Ca 2+ ) stimulates src and fyn , which activate PI3K via

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Activation Assay

    PI3K inhibition blocks the induction of involucrin and transglutaminase expression by calcium. Cultured human keratinocytes were preincubated with 10 mM LY294002 (+) or vehicle DMSO (–) for 1 h and then treated with 1.2 mM calcium for 24 h. The

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: PI3K inhibition blocks the induction of involucrin and transglutaminase expression by calcium. Cultured human keratinocytes were preincubated with 10 mM LY294002 (+) or vehicle DMSO (–) for 1 h and then treated with 1.2 mM calcium for 24 h. The

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Inhibition, Expressing, Cell Culture

    PIP 3 stimulates PLC-γ1 activity, and the binding of PIP 3 to PLC-γ1 is induced by calcium. (A). Human keratinocytes were cultured in KGM with 0.03 mM calcium. The total cell lysate was isolated with the cell lysis buffer containing 50 mM

    Journal:

    Article Title: Calcium-induced Human Keratinocyte Differentiation Requires src- and fyn-mediated Phosphatidylinositol 3-Kinase-dependent Activation of Phospholipase C-?1

    doi: 10.1091/mbc.E05-02-0109

    Figure Lengend Snippet: PIP 3 stimulates PLC-γ1 activity, and the binding of PIP 3 to PLC-γ1 is induced by calcium. (A). Human keratinocytes were cultured in KGM with 0.03 mM calcium. The total cell lysate was isolated with the cell lysis buffer containing 50 mM

    Article Snippet: Human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM; Cambrex Bio Science Walkersville, Walkersville, MD) as described previously ( ).

    Techniques: Planar Chromatography, Activity Assay, Binding Assay, Cell Culture, Isolation, Lysis

    Expression of miR-16 and miR-451 in the exosomes isolated from the different techniques and volumes of serum. ddPCR was used to measure the absolute concentration of miR-16 and miR-451 in the exosomes isolated with miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) using the different volumes of human serum. The values in the graph are the mean concentration (copies/μl) ± SEM (n = 3), *p≤0.05.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Expression of miR-16 and miR-451 in the exosomes isolated from the different techniques and volumes of serum. ddPCR was used to measure the absolute concentration of miR-16 and miR-451 in the exosomes isolated with miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) using the different volumes of human serum. The values in the graph are the mean concentration (copies/μl) ± SEM (n = 3), *p≤0.05.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Expressing, Isolation, Concentration Assay

    Size distribution of the exosomes isolated from pooled human serum using the different techniques and serum volumes. For each sample volume (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and technique, the number of particles per a specific particle size (nm) was measured using the ZetaView for NTA. Each graph represents quadratic interpolation of the mean number of particles isolated by each technique (n = 3). Data from the commercial kits miRCURY (blue), ExoQuick (green), and TEIR (red) are graphed on the left y-axis, while the UC (yellow) data, being at a significantly lower magnitude, are mapped on the right y-axis.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Size distribution of the exosomes isolated from pooled human serum using the different techniques and serum volumes. For each sample volume (5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl) and technique, the number of particles per a specific particle size (nm) was measured using the ZetaView for NTA. Each graph represents quadratic interpolation of the mean number of particles isolated by each technique (n = 3). Data from the commercial kits miRCURY (blue), ExoQuick (green), and TEIR (red) are graphed on the left y-axis, while the UC (yellow) data, being at a significantly lower magnitude, are mapped on the right y-axis.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    Correlation between the volume of serum and the zeta potential of the isolated solutions. The data in this graph are the mean values of three experimental replicates (n = 3) ± SEM. (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Correlation between the volume of serum and the zeta potential of the isolated solutions. The data in this graph are the mean values of three experimental replicates (n = 3) ± SEM. (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    Characterization of exRNA extracted from exosomes isolated using the four techniques. exRNA quality was evaluated using the Agilent Bioanalyzer with RNA 6000 Pico kit for the exosomes extracted using the different isolation techniques and serum volumes. The y-axis represents fluorescence, and the x-axis is the size of the RNA, measured in nucleotides (nt). (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Characterization of exRNA extracted from exosomes isolated using the four techniques. exRNA quality was evaluated using the Agilent Bioanalyzer with RNA 6000 Pico kit for the exosomes extracted using the different isolation techniques and serum volumes. The y-axis represents fluorescence, and the x-axis is the size of the RNA, measured in nucleotides (nt). (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation, Fluorescence

    Zeta potential measurements for individual serum exosomes isolated from the different techniques and serum volumes. Using the ZetaView instrument, the zeta potential (mV) was measured for the exosomes isolated from individ ual human serum samples using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) with starting volumes of 1 ml, 500 μl, and 100 μl. The data in this graph are the mean values of the zeta potential (n = 3) ± SEM, *p≤0.05.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Zeta potential measurements for individual serum exosomes isolated from the different techniques and serum volumes. Using the ZetaView instrument, the zeta potential (mV) was measured for the exosomes isolated from individ ual human serum samples using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) with starting volumes of 1 ml, 500 μl, and 100 μl. The data in this graph are the mean values of the zeta potential (n = 3) ± SEM, *p≤0.05.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    ZetaView measurements of the exosomes extracted from the six serum starting volumes of pooled human serum using the 4 different isolation techniques. NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl of human serum. The data in this graph are the mean values of three experimental replicates (±SEM), *p≤0.05.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: ZetaView measurements of the exosomes extracted from the six serum starting volumes of pooled human serum using the 4 different isolation techniques. NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 5 ml, 1 ml, 500 μl, 250 μl, 100 μl, and 50 μl of human serum. The data in this graph are the mean values of three experimental replicates (±SEM), *p≤0.05.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    Quantity of exRNA extracted from exosomes isolated using the different techniques and serum volumes. exRNA concentration was measured using the Agilent Bioanalyzer with RNA 6000 Pico kit for the exosomes extracted using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) with the six different serum volumes. Total amount of exRNA in each isolation was compared between different techniques. The data in this graph are the mean RNA amount (ng) of three experimental replicates (n = 3) ± SEM.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Quantity of exRNA extracted from exosomes isolated using the different techniques and serum volumes. exRNA concentration was measured using the Agilent Bioanalyzer with RNA 6000 Pico kit for the exosomes extracted using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) with the six different serum volumes. Total amount of exRNA in each isolation was compared between different techniques. The data in this graph are the mean RNA amount (ng) of three experimental replicates (n = 3) ± SEM.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation, Concentration Assay

    Zeta potential measurements for exosomes isolated from the different techniques and serum volumes. Using the ZetaView instrument, the zeta potential (mV) was measured for the exosomes isolated with miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 5 ml, 1 ml, 500 μl, 250 μl, and 100 μl of human serum. The data in this graph are the mean values of the zeta potential ± SEM (n = 3)

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Zeta potential measurements for exosomes isolated from the different techniques and serum volumes. Using the ZetaView instrument, the zeta potential (mV) was measured for the exosomes isolated with miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 5 ml, 1 ml, 500 μl, 250 μl, and 100 μl of human serum. The data in this graph are the mean values of the zeta potential ± SEM (n = 3)

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    Correlation between the volume of serum and the total number of particles isolated. The relationship between the volume of serum and the total number of particles was linear for all four isolation techniques: (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC. The data in this graph represent the average number of isolated particles ± SEM (n = 3).

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: Correlation between the volume of serum and the total number of particles isolated. The relationship between the volume of serum and the total number of particles was linear for all four isolation techniques: (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC. The data in this graph represent the average number of isolated particles ± SEM (n = 3).

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    ZetaView measurements of the exosomes extracted from three different serum starting volumes of six individual human donors samples using the 4 different isolation techniques. NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 1 ml, 500 μl, and 100 μl of human serum. The data in this graph are the mean values of the six individual human samples (±SEM), *p≤0.05.

    Journal: PLoS ONE

    Article Title: A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents

    doi: 10.1371/journal.pone.0170628

    Figure Lengend Snippet: ZetaView measurements of the exosomes extracted from three different serum starting volumes of six individual human donors samples using the 4 different isolation techniques. NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 1 ml, 500 μl, and 100 μl of human serum. The data in this graph are the mean values of the six individual human samples (±SEM), *p≤0.05.

    Article Snippet: We used three exosome isolation kits for this study: miRCURYTM exosome isolation kit-serum and plasma (miRCURY) (Exiqon, Woburn, MA), ExoQuickTM Serum Exosome Precipitation Solution (ExoQuick) (System Biosciences, Mountain view, CA), and Total Exosome Isolation Reagent for serum (TEIR) (Life Technologies, Carlsbad, CA).

    Techniques: Isolation

    Binding of cyclin D3 to pRb in vitro. The schematic in the upper inset shows the structure of the GST-Rb fusion proteins used and illustrates the A/B pocket region and the carboxy-terminal region of Rb. The lower inset shows schematic representations

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: Binding of cyclin D3 to pRb in vitro. The schematic in the upper inset shows the structure of the GST-Rb fusion proteins used and illustrates the A/B pocket region and the carboxy-terminal region of Rb. The lower inset shows schematic representations

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Binding Assay, In Vitro

    Kinetics of cyclin D3 or cyclin D3(T283A) protein turnover. C2 ( Rb +/+ ) or CC42 ( Rb − / − ) myoblasts were infected with retroviruses encoding wild-type Flag-CycD3 or Flag-CycD3(T283A). The infected cells were cultured in GM

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: Kinetics of cyclin D3 or cyclin D3(T283A) protein turnover. C2 ( Rb +/+ ) or CC42 ( Rb − / − ) myoblasts were infected with retroviruses encoding wild-type Flag-CycD3 or Flag-CycD3(T283A). The infected cells were cultured in GM

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Infection, Cell Culture

    The nuclear accumulation of cyclin D3 is restored in Rb − / − myocytes by expression of exogenous pRb, inhibition of GSK-3β activity, or inhibition of nuclear export. (A) C2 or CC42 myoblasts plated on glass coverslips were exposed

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: The nuclear accumulation of cyclin D3 is restored in Rb − / − myocytes by expression of exogenous pRb, inhibition of GSK-3β activity, or inhibition of nuclear export. (A) C2 or CC42 myoblasts plated on glass coverslips were exposed

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Expressing, Inhibition, Activity Assay

    Ectopic expression of pRb restores normal levels of cyclin D3 protein in differentiating Rb − / − myocytes. (A) Proliferating CC42 myoblasts ( Rb − / − ) were infected with Adeno-Rb or Adeno-β-gal and cultured in GM or

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: Ectopic expression of pRb restores normal levels of cyclin D3 protein in differentiating Rb − / − myocytes. (A) Proliferating CC42 myoblasts ( Rb − / − ) were infected with Adeno-Rb or Adeno-β-gal and cultured in GM or

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Expressing, Infection, Cell Culture

    The effect of ectopic expression of stabilized cyclin D3 mutants on differentiation of Rb +/+ myoblasts. (A) C2 myoblasts were infected with pBABEpuro control virus or viruses expressing the cyclin D3(T283A) or the cyclin D3-mLXCXE/dl(250-272)

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: The effect of ectopic expression of stabilized cyclin D3 mutants on differentiation of Rb +/+ myoblasts. (A) C2 myoblasts were infected with pBABEpuro control virus or viruses expressing the cyclin D3(T283A) or the cyclin D3-mLXCXE/dl(250-272)

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Expressing, Infection

    Lithium chloride (LiCl) enhances terminal differentiation of both Rb +/+ and Rb − / − myocytes, but ectopic expression of a stabilized cyclin D3 mutant in Rb − / − myocytes is not sufficient to reproduce the promyogenic

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: Lithium chloride (LiCl) enhances terminal differentiation of both Rb +/+ and Rb − / − myocytes, but ectopic expression of a stabilized cyclin D3 mutant in Rb − / − myocytes is not sufficient to reproduce the promyogenic

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Expressing, Mutagenesis

    Cyclin D3 deficiency results in defects in MyoD-induced myogenic conversion in MEFs. Wild-type and cyclin D3 −/− MEFs were infected with Adeno-MyoD (+Ad-MyoD) at an MOI of 500 or mock infected (−Ad-MyoD). Cells were shifted

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: Cyclin D3 deficiency results in defects in MyoD-induced myogenic conversion in MEFs. Wild-type and cyclin D3 −/− MEFs were infected with Adeno-MyoD (+Ad-MyoD) at an MOI of 500 or mock infected (−Ad-MyoD). Cells were shifted

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Infection

    GSK-3β phosphorylates cyclin D3 on Thr-283. (A) Glutathione-Sepharose-bound GST or GST-D3 (1 μg) was incubated with human recombinant GSK-3β in a kinase assay mixture containing [γ- 32 P]ATP. Proteins were resolved by SDS-PAGE,

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: GSK-3β phosphorylates cyclin D3 on Thr-283. (A) Glutathione-Sepharose-bound GST or GST-D3 (1 μg) was incubated with human recombinant GSK-3β in a kinase assay mixture containing [γ- 32 P]ATP. Proteins were resolved by SDS-PAGE,

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Incubation, Recombinant, Kinase Assay, SDS Page

    Thr-283 is required for cyclin D3 ubiquitination. C2 ( Rb +/+ ) or CC42 ( Rb − / − ) proliferating myoblasts were transfected with expression vectors encoding wild-type Flag-CycD3 or Flag-CycD3(T283A) together with an expression

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: Thr-283 is required for cyclin D3 ubiquitination. C2 ( Rb +/+ ) or CC42 ( Rb − / − ) proliferating myoblasts were transfected with expression vectors encoding wild-type Flag-CycD3 or Flag-CycD3(T283A) together with an expression

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Transfection, Expressing

    pRb inhibits the association between cyclin D3 and GSK-3β, thus preventing cyclin D3 phosphorylation by GSK-3β. (A) Insect Sf9 cells were infected with baculoviruses encoding cyclin D3, GSK-3β, or Flag-Rb. Sf9 cell extract containing

    Journal:

    Article Title: pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

    doi: 10.1128/MCB.02199-06

    Figure Lengend Snippet: pRb inhibits the association between cyclin D3 and GSK-3β, thus preventing cyclin D3 phosphorylation by GSK-3β. (A) Insect Sf9 cells were infected with baculoviruses encoding cyclin D3, GSK-3β, or Flag-Rb. Sf9 cell extract containing

    Article Snippet: Coverslips were incubated for 30 min with PBS containing 20% goat serum and then incubated for 2 h with the following antibodies in PBS-5% goat serum: anti-cyclin D3 (C-16; Santa Cruz) used at 2 μg/ml; anti-Rb (G3-245; Pharmingen) used at 10 μg/ml; and anti-GSK-3β (610202; BD Transduction Laboratories) used at 2.5 μg/ml.

    Techniques: Infection

    ChIP analysis of estrogen target genes. (A) Soluble chromatin was prepared from MCF7 cells not treated or treated with E2 for 45 min. Immunoprecipitation was performed with antibodies against RIZ1, dimethylated H3-K9, dimethylated H3-K4, acetylated H3-K9,

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: ChIP analysis of estrogen target genes. (A) Soluble chromatin was prepared from MCF7 cells not treated or treated with E2 for 45 min. Immunoprecipitation was performed with antibodies against RIZ1, dimethylated H3-K9, dimethylated H3-K4, acetylated H3-K9,

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation

    RIZ1 interacts with HAT-class coactivators. (A) RIZ1 enhances the coactivator functions of members of the SRC-1 (p160) family. The indicated amounts of RIZ1, ERE-tk-CAT, ERα, and SRC-1 or GRIP-1 expression vectors were cotransfected into CV-1

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: RIZ1 interacts with HAT-class coactivators. (A) RIZ1 enhances the coactivator functions of members of the SRC-1 (p160) family. The indicated amounts of RIZ1, ERE-tk-CAT, ERα, and SRC-1 or GRIP-1 expression vectors were cotransfected into CV-1

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: HAT Assay, Expressing

    Uterine and vaginal responses to female sex steroid hormones in RIZ1 mutant mice. Eight-week-old females with the indicated genotypes were OVXed and then treated for 3 days with E2 or the vehicle (V) alone from days 15 to 17 post-OVX. Uterine and vaginal

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Uterine and vaginal responses to female sex steroid hormones in RIZ1 mutant mice. Eight-week-old females with the indicated genotypes were OVXed and then treated for 3 days with E2 or the vehicle (V) alone from days 15 to 17 post-OVX. Uterine and vaginal

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Mutagenesis, Mouse Assay

    Regulation of RIZ1 methyltransferase activity by estrogen. The same amounts of nuclear extracts (500 μg) from E2-treated or control MCF-7 cells or from AdRIZ1-infected cells were immunoprecipitated with preimmune serum or RIZ1 serum anti-KG7.1S.

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Regulation of RIZ1 methyltransferase activity by estrogen. The same amounts of nuclear extracts (500 μg) from E2-treated or control MCF-7 cells or from AdRIZ1-infected cells were immunoprecipitated with preimmune serum or RIZ1 serum anti-KG7.1S.

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Activity Assay, Infection, Immunoprecipitation

    Normal steroid hormone response in RIZ1 -deficient male mice.

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Normal steroid hormone response in RIZ1 -deficient male mice.

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Mouse Assay

    Model of RIZ1 in estrogen action. In the absence of estrogen, RIZ1 sits on estrogen target genes through direct protein-DNA interaction and represses transcription through methylation of H3-K9. In the presence of estrogen, ER binds to RIZ1 and switches

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Model of RIZ1 in estrogen action. In the absence of estrogen, RIZ1 sits on estrogen target genes through direct protein-DNA interaction and represses transcription through methylation of H3-K9. In the presence of estrogen, ER binds to RIZ1 and switches

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Methylation

    RIZ1 is a selective coactivator of ER and PR. (A) Promoter reporter analysis. The indicated amounts of RIZ1 and various NHR expression vectors were cotransfected with a reporter construct containing appropriate hormone receptor response element into CV-1

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: RIZ1 is a selective coactivator of ER and PR. (A) Promoter reporter analysis. The indicated amounts of RIZ1 and various NHR expression vectors were cotransfected with a reporter construct containing appropriate hormone receptor response element into CV-1

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Expressing, Construct

    Impaired regulation of uterine PR expression by E2 in RIZ1 mutant mice. Uteri were collected and immunohistochemically analyzed for PR expression. (A) Estrogen-treated uteri (top panel, low magnification; bottom panel, high magnification). Randomly selected

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Impaired regulation of uterine PR expression by E2 in RIZ1 mutant mice. Uteri were collected and immunohistochemically analyzed for PR expression. (A) Estrogen-treated uteri (top panel, low magnification; bottom panel, high magnification). Randomly selected

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Expressing, Mutagenesis, Mouse Assay

    Mammopoiesis in RIZ1 deficient mice. Whole mounts of the fourth mammary gland of mice with the indicated genotypes were prepared and stained as described in Materials and Methods. (A and B) Seven-week-old virgin mice; (C and D) mice pregnant for the first

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Mammopoiesis in RIZ1 deficient mice. Whole mounts of the fourth mammary gland of mice with the indicated genotypes were prepared and stained as described in Materials and Methods. (A and B) Seven-week-old virgin mice; (C and D) mice pregnant for the first

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Mouse Assay, Staining

    Knockdown of RIZ1 by siRNA affects expression and methylation of pS2 gene. (A) Western blot analysis of equal amounts of nuclear extracts derived from siRNA and control vector-transfected MCF7 cells. Antibodies used are indicated on the left side of the

    Journal:

    Article Title: A Histone Methyltransferase Is Required for Maximal Response to Female Sex Hormones

    doi: 10.1128/MCB.24.16.7032-7042.2004

    Figure Lengend Snippet: Knockdown of RIZ1 by siRNA affects expression and methylation of pS2 gene. (A) Western blot analysis of equal amounts of nuclear extracts derived from siRNA and control vector-transfected MCF7 cells. Antibodies used are indicated on the left side of the

    Article Snippet: Western blot analyses with anti-RIZ1 rabbit serum (Abcam) and anti-PARP (poly-ADP-ribose polymerase) antibody (Santa Cruz Biotech) were performed with nuclear extracts derived from cells at 2 days posttransfection.

    Techniques: Expressing, Methylation, Western Blot, Derivative Assay, Plasmid Preparation, Transfection

    Isotyping of the Anti-CHO-HCP Antibodies in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Isotyping of the Anti-CHO-HCP Antibodies in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Isotyping of the Anti-CHO-HCP Antibodies in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Isotyping of the Anti-CHO-HCP Antibodies in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Confirmation of the Specificity of Anti-CHO-HCP Antibody Isotypes in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the Specificity of Anti-CHO-HCP Antibody Isotypes in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Confirmation of the Specificity of the Positive Anti-CHO-HCP Reactivity in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the Specificity of the Positive Anti-CHO-HCP Reactivity in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Confirmation of the Specificity of the Positive Anti-CHO-HCP Reactivity in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the Specificity of the Positive Anti-CHO-HCP Reactivity in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Isotyping of the Anti-CHO-HCP Antibodies in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Isotyping of the Anti-CHO-HCP Antibodies in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Prevalence of anti-CHO-HCP reactivity in normal human serum ( HS ) samples. A validated ELISA was used to detect the reactivity to the drug process-specific CHO-HCP in 83 serum samples collected from individuals with no known prior exposure to therapeutic

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Prevalence of anti-CHO-HCP reactivity in normal human serum ( HS ) samples. A validated ELISA was used to detect the reactivity to the drug process-specific CHO-HCP in 83 serum samples collected from individuals with no known prior exposure to therapeutic

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques: Enzyme-linked Immunosorbent Assay

    Confirmation of the specificity of the IgG4 type anti-CHO-HCP antibody in normal human serum ( HS ) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml,

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the specificity of the IgG4 type anti-CHO-HCP antibody in normal human serum ( HS ) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml,

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques: Inhibition, Incubation, Purification

    Confirmation of the positive reactivity to CHO-HCP in normal human serum ( HS ) samples—competitive inhibition specificity testing. Normal human serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml,

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the positive reactivity to CHO-HCP in normal human serum ( HS ) samples—competitive inhibition specificity testing. Normal human serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10, 100, and 500 μg/ml,

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques: Inhibition, Incubation, Purification

    Confirmation of the specificity of the anti-CHO-HCP antibody isotypes in normal human serum ( HS ) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10/ml, 100, and

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the specificity of the anti-CHO-HCP antibody isotypes in normal human serum ( HS ) samples—competitive inhibition specificity testing. Serum samples were pre-incubated with purified CHO-HCP at concentrations of 0, 10/ml, 100, and

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques: Inhibition, Incubation, Purification

    Confirmation of the Specificity of Anti-CHO-HCP Antibody Isotypes in Normal Human Serum Samples

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Confirmation of the Specificity of Anti-CHO-HCP Antibody Isotypes in Normal Human Serum Samples

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques:

    Identification of the IgG4 type reactivity in anti-CHO-HCP-positive serum samples using the biotin mouse anti-human IgG4 detector. IgG4 isotypic cut point OD is defined as two times the mean of the negative sample OD. Samples with OD values greater than

    Journal:

    Article Title: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

    doi: 10.1208/s12248-009-9165-5

    Figure Lengend Snippet: Identification of the IgG4 type reactivity in anti-CHO-HCP-positive serum samples using the biotin mouse anti-human IgG4 detector. IgG4 isotypic cut point OD is defined as two times the mean of the negative sample OD. Samples with OD values greater than

    Article Snippet: A pool of sheep anti-CHO-HCP serum (Wyeth Research) was used as the assay positive control.

    Techniques: