Journal: Investigative ophthalmology & visual science

Article Title: The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet's Membrane Components.

doi: 10.1167/iovs.16-20087

Figure Lengend Snippet: FIGURE 4. Phase-contrast images and cell-surface marker expression of the cHCECs used in Figure 5. (A) Phase-contrast images and FACS analysis of the mature cHCEC SP purified by MACS CD44-negative selection. FACS analysis was performed as follows: cHCECs were detached from the culture dish and analyzed with regard to the expression of CD166, CD24, CD44, CD105, and CD26 with a FACS Canto II flow cytometer as described in Materials and Methods. After gating for CD166þCD24 (R1), the following 3 SPs were defined: CD166þCD24CD44/þCD105þ/ SP (gate 1 ¼ G1), CD166þCD24CD44þþCD105þ SP (G2), and CD166þCD24CD44þþþCD105þ SP (G3). (B) The cHCECs shown in (A) were stained with ZO-1 and Naþ/Kþ ATPase. Nuclei were stained with DAPI. (C) Phase-contrast images and FACS analysis of the EMT-phenotype SP. (D) The cHCECs shown in (C) were stained with ZO-1 and Naþ/Kþ ATPase. Nuclei were stained with DAPI.

Article Snippet: The HCECs were detached with TrypLE Select as described above, and the CD44 HCEC SP (the effector SP) was isolated using anti-human CD44 MicroBeads and the depl05 program of an autoMACS Pro Separator (Miltenyi Biotec, Bergisch Downloaded From: https://iovs.arvojournals.org/ on 08/15/2018 Gladbach, Germany).

Techniques: Marker, Expressing, Selection, Cytometry, Staining

Journal: Investigative ophthalmology & visual science

Article Title: The Different Binding Properties of Cultured Human Corneal Endothelial Cell Subpopulations to Descemet's Membrane Components.

doi: 10.1167/iovs.16-20087

Figure Lengend Snippet: FIGURE 6. Expression of the integrin alpha (a) subunits on cHCEC SPs. The surface expression of integrin a2, a3, a6, and CD44 on mature cHCEC SPs and the EMT-phenotype SP were analyzed via the use of a FACSCanto II Flow Cytometry Analyzer System (BD Biosciences).

Article Snippet: The HCECs were detached with TrypLE Select as described above, and the CD44 HCEC SP (the effector SP) was isolated using anti-human CD44 MicroBeads and the depl05 program of an autoMACS Pro Separator (Miltenyi Biotec, Bergisch Downloaded From: https://iovs.arvojournals.org/ on 08/15/2018 Gladbach, Germany).

Techniques: Expressing, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 1. S180A mutation in CD44 prevents CS addition and results in constitutive HA binding in Jurkat cells. A, CD44 expression and HA binding were analyzed by flow cytometry in transfected Jurkat cells either unstimulated or stimulated for 8 h with PMA. The two left panels show expression levels of CD44, detected using Alexa 488 conjugated IM7, whereas the two right panels show binding to Fl-HA. B, CD44 immuno- precipitated from sodium [35S]sulfate-labeled cells grown in the presence or absence of -D-xyloside and resolved by SDS-PAGE. C, Relative quan- titation of sulfate incorporation by CD44 before and after digestion with chondroitinase ABC (ABC) or heparitinase (Hep) as measured by densi- tometry. Sulfate incorporation by untreated wild-type CD44 was set to 100% after adjustment for the level of CD44. Data are shown as the mean SD of three experiments with significance determined by the Stu- dent’s t test (, p 0.001). D, Detection of CS on immunoprecipitated CD44 by Western blotting with the anti-CS mAb 2B6. CD44 loading levels were determined by blotting with the mAb 3G12.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Binding Assay, Expressing, Cytometry, Transfection, Labeling, SDS Page, Immunoprecipitation, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 2. Cell death is preferentially induced in TCR- or PMA-stim- ulated Jurkat cells expressing a high HA binding form of CD44. A, Time course showing percentage of live cells after stimulation with the anti-TCR mAb C305. Data are shown as the mean SD of three experiments and were normalized by setting the number of live events in the untreated samples to 100%. B, Representative experiment showing the percentage of cells that were unstained (viable), single positive for Annexin V, or double positive for both Annexin V and PI following anti-TCR stimu- lation. C, Time course of cell viability during PMA stimulation as de- termined by lack of Annexin V-FITC and PI staining. Data are shown as the mean SD of three experiments. D, Same as B, except cells were stimulated with PMA.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Expressing, Binding Assay, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 3. CD44 mAbs can induce or prevent cell death of transfected Jurkat cells during PMA stimulation. A, Representative experiment show- ing the percentage of cells that were viable, single positive for Annexin V, or double positive for both Annexin V and PI following incubation with Hermes-1 or Hermes-3 for 20 min and cross-linking with secondary Ab for 16 h. B, Same as A, except unstimulated or PMA stimulated cells were grown in AIMV serum-free media before coincubation of Hermes-1 and secondary Ab for 8 h. C, Graph showing percentage of viable cells fol- lowing PMA stimulation for 16 h in the presence or absence of the HA blocking anti-CD44 mAb Hermes-1 or the nonblocking anti-CD44 mAb Hermes-3. Data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.01, , p 0.001).

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Transfection, Incubation, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 4. Enhanced AICD in Jurkat transfectants is dependent upon HA binding by CD44. A, Graph showing cell viability in S180A-CD44 cells following PMA stimulation for 16 h in RPMI 1640 supplemented with 10% FCS or in RPMI 1640 with or without 5 g/ml HA. Hyaluron- idase (HA’ase) treatment was done for 30 min before PMA stimulation. Data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.05, , p 0.01, , p 0.001). B, Cell viability is shown following PMA stimulation for 16 h in AIMV serum-free media with various concentrations of HA. The HA- blocking anti-CD44 mAb Hermes-1 was added to some samples. C, The mean fluorescence intensity (MFI) for CD44 and S180A-CD44 expression (top panel) and Fl-HA binding (middle panel) at various time points during PMA stimulation is shown. The bottom panel shows the relationship be- tween Fl-HA binding and the level of CD44 expression for CD44 and S180A-CD44. Data in B and C are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Binding Assay, Blocking Assay, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 5. The size of HA affects its ability to induce cell death in PMA-stimulated cells. A, Visualization of high, intermediate (int), or low molecular mass HA by silver staining samples run on a 15% acrylamide gel. Bromophenol blue (BPB) was used as a marker dye. B, Competition assay between Fl-HA and increasing amounts of unlabeled high, interme- diate, and low molecular mass HA in S180A-CD44 cells. C, Cell viability is shown in S180A-CD44 cells following PMA stimulation for 16 h in AIMV serum-free media with various concentrations of high, intermediate, or low molecular mass HA. The HA-blocking anti-CD44 mAb Hermes-1 was added to some samples. Data in B and C are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Silver Staining, Acrylamide Gel Assay, Marker, Competitive Binding Assay, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 6. HA can rapidly induce cell death in CD44-expressing cells after PMA activation. A, Graph showing cell viability in S180A-CD44 cells suspended in AIMV media and stimulated with PMA in the presence or absence of 500 ng/ml HA. Cells were initially stimulated for 12 h, then washed twice and incubated for an additional 4 h in the presence of PMA and/or HA. Data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.05, , p 0.001). B, Time course of cell viability following the addition of HA to S180A-CD44 cells prestimulated for 12 h with PMA. Hermes-1 mAb was added 20 min before the addition of HA for one sample set. Data are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 7. HA-induced cell death does not occur via the Fas/FasL pathway. A, Cell viability was assessed in unstimulated cells incubated with the anti-Fas mAb 7C11 for various times. Data are shown as the mean SD of three experiments. B, Fas expression as determined by flow cytometry in cells cultured in 10% FCS and either stimulated with PMA for 8 h or left untreated (NT). C, FasL mRNA expression in AIMV-cultured S180A-CD44 cells as determined by semiquantitative PCR following PMA stimulation in the presence or absence of HA. -actin was used as a loading control. D, Cell viability of S180A-CD44 transfectants following the addition of HA for 2 h to cells prestimulated with PMA. Cells were preincubated for 20 min with the Fas-blocking mAb ZB4 or the FasL-blocking mAb NOK-1 before the addition of HA. E, Cell viability of S180A-CD44 cells activated for 16 h with immobilized anti-CD3 mAb OKT3 in the presence of various combinations of HA, ZB4, or Hermes-1. F, Western blot analysis of caspase 8 activation following the addition of HA to PMA-stimulated S180A-CD44 cells. Incubation of unstimulated cells with the anti-Fas mAb 7C11 for 2 h was used as a positive control. G, Same as D, except cells were preincubated for 20 min with 25 or 50 M of the pan-caspase inhibitor z-VAD-fmk. As a control, unstimulated cells were incubated with z-VAD-fmk before the addition of the 7C11 mAb. For D, E, and G data are shown as the mean SD of three experiments with significance determined by the Student’s t test (, p 0.01, , p 0.001).

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Incubation, Expressing, Cytometry, Cell Culture, Control, Blocking Assay, Western Blot, Activation Assay, Positive Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 8. HA-induced cell death does not occur via the mitochon- drial pathway. A, Cell viability of unstimulated Jurkat transfectants in- cubated with staurosporine for 1–3 h (left panel) or incubated in RPMI 1640 without FCS for 1–3 days (right panel). B, Time course of mito- chondrial depolarization in S180A-CD44 cells. PMA-stimulated cells were incubated with either HA or Hermes-1 mAb and goat anti-rat Ab (2°) and then labeled with the mitochondrial membrane-specific dye JC-1. Incubation of unstimulated cells with the 7C11 mAb was used as a positive control. C, Same as B, except cells were analyzed after 2 h with the membrane dye DiOC6(3) and the percentage of live depolar- ized cells (PI negative, DiOC6(3) low) is shown. D, Western blot anal- ysis of caspase 3 activation following the addition of HA to PMA- stimulated S180A-CD44 cells. Data from A, B, and C are shown as the mean SD of three experiments.

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Incubation, Labeling, Membrane, Positive Control, Western Blot, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Hyaluronan induces cell death in activated T cells through CD44.

doi: 10.4049/jimmunol.181.10.7044

Figure Lengend Snippet: FIGURE 10. AICD in ex vivo activated murine splenic T cells is en- hanced by the presence of HA. A, Representative experiment showing the percentage of live day 6 splenic T cells from wild-type (WT) and CD44 knockout (KO) mice following reactivation with immobilized anti-CD3 mAb 145-2C11 for 24 h (see Materials and Methods for details). Some samples were incubated with 500 ng/ml HA or with both immobilized anti-CD3 and anti-CD44 (IM7). The mean of three experiments is shown. B, Analysis of CD44 expression and Fl-HA binding of day 6 activated splenic T cells either unstimulated (thin line) or restimulated (thick line) for 24 h. The cells alone negative control (shaded) is also shown. C, Analysis of PS exposure and Fl-HA binding in day 6 wild-type splenic T cells. After restimulation for 24 h with 2.5 g/ml of immobilized anti-CD3 mAb, cells were incubated with Fl-HA for 30 min at 37°C, labeled with Annexin V-PE on ice, and then analyzed by flow cytometry. Live cells were divided into non-, low-, and high-HA binding populations and analyzed for levels of Annexin V-PE binding. The percent of cells positive for Annexin V within each population is indicated. Mean fluorescence intensity (MFI) was nor- malized between experiments by setting the intensity of the non-HA bind- ing population to 1, and data are shown as the mean SD of three ex- periments with significance determined by the Student’s t test (, p 0.05). D, Graph showing the relative decrease in cell viability in day 6 T cells incubated in the presence of 1 g/ml of the Fas blocking mAb MFL3 for 24 h on immobilized anti-CD3 mAb. HA at 500 ng/ml was added to the cells for 0.5 h, 2 h, or 24 h. To normalize between experiments, the per- centage of loss of cell viability between cells stimulated in the absence vs the presence of HA is shown. Data are the mean SEM of four experi- ments with pools of two mice per experiment. Significance (, p 0.01) is shown compared with CD44 knockout (KO) cells. E, Graph showing the

Article Snippet: Purified rat anti-human/mouse CD44 mAb IM7.8.1 (ATCC no. TIB-235) was conjugated to Alexa 488 (Molecular Probes) or coupled to cyanogen bromideactivated Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions.

Techniques: Ex Vivo, Knock-Out, Incubation, Expressing, Binding Assay, Negative Control, Labeling, Cytometry, Blocking Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: The effect of statins on viability and growth of ( a ) stem ADMSC and non-cancerous HEK 293 cells and ( b ) cancer MiaPaCa-2 cells. ( a ) ADMSC—human adipose-derived mesenchymal stem cells, HEK 293—human embryonic kidney cells, exposure to statins—24 h, concentrations 0—100 µM, control—methanol, ( b ) previously published data , MiaPaCa-2—pancreatic cancer cells, exposure to statins—24 h, concentrations 0—40 µM, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of the effect of statins on the growth and viability of pancreatic cancer MiaPaCa-2 cells, non-cancerous HEK 293 cells, and ADMSC stem cells. Concentration of statins—20 µM, Time—exposure to statins—24, 48, and 72 h, control—methanol.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Concentration Assay, Control

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on size and compactness of spheroids. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr—methanol treated spheroids, P—pravastatin, R—rosuvastatin, L—lovastatin, F—fluvastatin, A—atorvastatin, Pi—pitavastatin, C—cerivastatin, S—simvastatin. Statins were added once, after spheroid formation, 10 weeks ( a ) or 3.5 weeks ( b ) after inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Effect of statins on the spheroid formation. ( a ) ADMSC stem cells, ( b ) pancreatic cancer MiaPaCa-2 cells, concentration of statins—20 µM, Ctr methanol treated spheroids, P —pravastatin, R —rosuvastatin, L —lovastatin, F —fluvastatin, A —atorvastatin, Pi —pitavastatin, C —cerivastatin, S —simvastatin. Statins were added once, 24 h after cell inoculation. Experiment was carried out in biological dodecaplicates.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Comparison of expression changes between statin treated and control MiaPaCa-2 and ADMSC cells. Displayed are only the genes that are differentially expressed upon at least one statin treatment in at least one cell type, requiring |log 2 FC|> 1 and FDR < 0.05. Statins were administered at a concentration of 12 µM for 24 h. ( FC fold change, FDR false discovery rate, horizontal and vertical axes—changes in ADMSC and MiaPaCa-2 cells, respectively, upon respective treatment). The red dashed lines indicate two-fold change increase or decrease in the gene expression. The genes with at least two-fold up-regulation (resp. down-regulation) in ADMSC stem cells are displayed to the right (resp. left) of the dashed lines. Similarly, genes with at least two-fold up-regulation (resp. down-regulation) in cancer cells are displayed above (resp. below) of the dashed lines. For details about differentially regulated transcripts see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579 .

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Comparison, Expressing, Control, Concentration Assay, Gene Expression

Journal: Scientific Reports

Article Title: Highly variable biological effects of statins on cancer, non-cancer, and stem cells in vitro

doi: 10.1038/s41598-024-62615-w

Figure Lengend Snippet: Cellular pathways most significantly affected by statins in cancer and stem cells. The gene set enrichment analysis (GSEA) revealed the KEGG pathways most affected by statin treatment in ADMSC and MiaPaCa-2 cells. Displayed is the union of the top five most enriched pathways among the comparisons. (Statin concentration—12 µM, treatment time—24 h, p-value—GSEA p-value, gene ratio—fraction of KEGG pathway genes among differentially expressed genes). For details about differentially regulated transcripts, see the ArrayExpress database, accessions E-MTAB-3979, E-MTAB-11579.

Article Snippet: Human adipose-derived mesenchymal stem cells ADMSC (ATCC, Manassas, VA, PSC-500-011, LOT 70017032, positive specific staining for CD29, CD44, CD73, CD90, CD105, and CD166 and negative for CD14, CD31, CD34, and CD45) were cultured in mesenchymal stem cell basal medium (ATCC, Manassas, VA) supplemented with low serum mesenchymal stem cell growth kit for adipose- and umbilical-derived MSCs (ATCC, Manassas, VA).

Techniques: Concentration Assay

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Activation Assay, Staining, Western Blot, Inhibition

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Membrane, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Disruption, Confocal Microscopy, Staining, Fluorescence, Western Blot

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Binding Assay, Western Blot, Isolation, SDS-Gel, Electrophoresis, Incubation, Labeling, Fluorescence

Journal: Nature Communications

Article Title: Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition

doi: 10.1038/ncomms7139

Figure Lengend Snippet: ( a ) Schematic of human explant model to evaluate response of refractory human tissue to anticancer agents. Tumour biopsies were cut into ~200 mM-thick sections and cultured in microwells coated with tumour matrix and media supplemented with autologous serum. ( b ) Representative immunohistochemistry (IHC) of primary human breast tumour explants shows induction of CD44 and CD24 cell surface expression following 72 h treatment with docetaxel versus vehicle. × 40 Scale bar, 50 μm inset show higher magnification, × 100 ( c , d ) Graph shows quantification of CD44 and CD24 levels in the primary tumour explant studies, ( N =14 patients). Black and red points denote the protein levels measured by IHC score in a tumour explant in vehicle- and docetaxel-treated groups. Each number denotes a patient. The orange arrows denote patients who were taxane-treatment naive, whereas those denoted with black arrows received a taxane. ( e ) Representative IHC from explant culture shows effect of different drug treatments (3.4 μM docetaxel, 5.6 μM doxorubicin) on the expression of CD44 and cleaved (cl) caspase 3 in corresponding serial sections. Gem, gemcitabine. × 40 magnification Scale bar, 50 μm. Inset shows higher magnification × 100 ( f ) Graph shows the quantification of CD44 and cleaved caspase 3 expression in the explants treated with docetaxel ( n =9) or a combination of gemcitabine+carboplatin ( n =2). Data shows mean±s.e.m. ( g ) Schematic shows generation of drug-tolerant cells (DTCs) selected acutely using high-concentration docetaxel chemotherapy in vitro. Cells were cultured in 100 μM (~20X IC 50 ) docetaxel. Cells surviving by day 4 were quiescent and considered as drug-tolerant cells (DTCs). Growing out the DTCs over 35 days resulted in restoring parental properties. ( h ) Graph shows MTS cell viability analysis of parental cells and DTCs generated from of MDA-MB-231 breast cancer cells following incubation (48 h) with different tubulin-binding chemotherapeutics at indicated concentration range. ( i ) Confocal images show expression levels of CD44 and CD24 in parental cells and DTCs generated from MDA-MB-468s. Scale bar, 18 μm ( j ) The population percentage of CD44 Hi CD24 Hi cells in parental and DTCs generated from an array of luminal and basal breast cancer cell lines. Data shown are mean±s.e.m., n =3 ( P <0.01 other than T47D cells). ( k ) Representative FACS plot of CD44 and CD24 in MDA-MB-231 parent cells and DTC. ( l ) Graph shows quantification of CD44 Hi /CD24 Lo and CD44 Hi /CD24 Hi as % of total population of MDA-MB-231 parent cells and DTCs (Data shown are mean±s.e.m., n =8, ANOVA analysis * P <0.01, *** P <0.001).

Article Snippet: Following primary antibodies were used: anti human Ki-67 (rabbit polyclonal from Vector Laboratory, 1:600 dilution), anti-human cleaved caspase3 (rabbit polyclonal, clone D175, Cell Signaling Technology, Cambridge, MA, USA), Anti-human CD44 (Clone IM7), P-Hck Y410 (Cell Signaling Technology).

Techniques: Cell Culture, Immunohistochemistry, Expressing, Concentration Assay, In Vitro, Generated, Incubation, Binding Assay

Journal: Nature Communications

Article Title: Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition

doi: 10.1038/ncomms7139

Figure Lengend Snippet: ( a ) Schematic shows experimental design used to derive mathematical parameters of population dynamics. Treatment of MDA-MB-231 breast cancer cells with 25 nM docetaxel (DTX) for 24 h induces phenotype plasticity rather than providing a selection pressure. In parallel, starting cells with different permutations and combinations of CD24 and CD44 expression levels were used, and the expression of CD44CD24 was monitored over defined time points. ( b ) Population dynamics modelling derived from experimental data indicates temporal kinetics of breast cancer cells in distinct compartments over 5 days (CD44 Lo described as non-CSC). Left panel shows dynamics of distinct phenotypes under basal conditions, right panel demonstrates population dynamics under chemotherapy pressure. ( c ) Schematic shows subpopulation transition dynamics and predictive contribution of each population under chemotherapy pressure or basal state, saturated to equilibrium. Arrow weights denote prevalence of conversion. Loops indicate propensity to replicate or transition. ( d ) Treatment-naive 231-parental cells were sorted into CD44 Hi CD24 Hi , CD44 Hi CD24 Lo , CD44 Lo CD24 Hi and CD44 Lo CD24 Lo subpopulations, which were subsequently exposed to high-dose docetaxel (100 nM) for 48 h and re-analyzed by FACS for CD44 Hi CD24 Hi subset expressed as % of total population. ‘Basal’ denotes the change in % of CD44 Hi CD24 Hi subset in parental cells treated with vehicle. Data are mean±s.e.m. (ANOVA analysis, N =7, # P <0.05, * P <0.05 ** P <0.01 versus basal group). ( e ) Depletion of intrinsic CSC population with salinomycin (5 μM, 48 h) was confirmed by reduction of a CSC (CD44 Hi /CD24 Lo ) and enrichment of a non-CSC phenotype (CD44 Lo CD24 Hi ) expressed as fold change from vehicle-treated cells (error bars indicate s.e.m., N =5, * P <0.05 ** P <0.01). ( f ) Chemo-tolerant cells generated from parent (DTC) and salinomycin-selected (Sal-DTC) MDA-MB-231 cells were analyzed by FACS for CD44 Hi /CD24 Hi , and results are expressed as % of total population (Data shown are mean±s.e.m., n =8, ANOVA analysis *** P <0.001, NS, not significant). ( g ) Graph shows mean fluorescent intensity (MFI) from FACS analysis of CD44 and CD24 expression in MDA-MB-231-parent, -DTC, -Sal-DTC or in a population of -expanded (E)-DTC and -Sal-DTC, demonstrating a reversal to parental phenotype when the chemotolerant cells are expanded over time (Data shown are mean±s.e.m. n =5, * P <0.05 ** P <0.01). ( h ) Graph shows cell viability of each indicated population to docetaxel or doxorubicin, quantified by MTS cytotoxicity assay as % of viability in vehicle-treated control. All data shown are mean±s.e.m. from independent replicates (ANOVA analysis, n =8, *** P <0.001 versus parent cells).

Article Snippet: Following primary antibodies were used: anti human Ki-67 (rabbit polyclonal from Vector Laboratory, 1:600 dilution), anti-human cleaved caspase3 (rabbit polyclonal, clone D175, Cell Signaling Technology, Cambridge, MA, USA), Anti-human CD44 (Clone IM7), P-Hck Y410 (Cell Signaling Technology).

Techniques: Selection, Expressing, Derivative Assay, Generated, Cytotoxicity Assay, Control

Journal: Nature Communications

Article Title: Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition

doi: 10.1038/ncomms7139

Figure Lengend Snippet: ( a ) Graph shows quantification of activated SFK in MDA-MB231 treated with high concentration docetaxel post siRNA knockdown of CD44, CD24 or both genes in ( N =4, ANOVA ** P <0.01). ( b ) Hck was immunoprecipitated from DTCs or parent MDA-MB-231s cell lysates followed by western blotting for CD44 and CD24 antibodies IgG input included for control. IgG HC indicates heavy chain (HC) bands. ( c ) Co-IP of Hck was performed from cell lysate of parent or DTCs generated from MDA-MB-231 cells transfected with siRNA targeting CD24 and CD44 or a combination of both. Western blotting indicates Cav-1 scaffolding to Hck. ( d ) Representative confocal images demonstrates colocalization of CD44, CD24 and Hck to lipid raft-rich regions of the cell membrane in DTC derived from MDA-MB-231 cells. Scale bar, 5 μm ( e ) Confocal microscopy identifies Caveolin 1 (Cav-1) colocalizing with CD24, CD44 and Hck in the MDA-MB-231 DTCs. Scale bar, 5 μm ( f ) Subcellular localization of Hck in DTC generated following siRNA-knockdown Cav-1. β-Actin and PARP indicate loading controls of cytoplasmic and nuclear compartments, respectively. ( g ) Confocal microscopy was used to identify subcellular localization in the nuclear plane of phosphorylated Hck (pHck) in the MDA-MB-231 parent compared with DTCs (upper panel). Dual staining shows a pattern of nuclear and perinuclear localized pHck and Cav-1 in the DTCs. Scale bar, 8 μm. ( h ) Representative confocal images reveal the expression of APAF-1 (Green signal) in MDA-MB-231 DTCs treated with vehicle or RK20449 (1 μM) for 24 h, and counterstained with DAPI. Scale bar, 8 μm.

Article Snippet: Following primary antibodies were used: anti human Ki-67 (rabbit polyclonal from Vector Laboratory, 1:600 dilution), anti-human cleaved caspase3 (rabbit polyclonal, clone D175, Cell Signaling Technology, Cambridge, MA, USA), Anti-human CD44 (Clone IM7), P-Hck Y410 (Cell Signaling Technology).

Techniques: Concentration Assay, Knockdown, Immunoprecipitation, Western Blot, Control, Co-Immunoprecipitation Assay, Generated, Transfection, Scaffolding, Membrane, Derivative Assay, Confocal Microscopy, Staining, Expressing

Journal: Nature Communications

Article Title: Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition

doi: 10.1038/ncomms7139

Figure Lengend Snippet: ( a ) Schematic shows the selection of ‘induced’ CD44 Hi CD24 Hi phenotype from 4T1 mammary carcinoma cells sorted by FACS. Cells that emerge de novo with a CD44 Hi CD24 Hi phenotype following chemotherapy treatment (50 nM docetaxel, 24 h) (~2.2% of the population that was not originally present in the parent population were considered an ‘induced’ subset. ( b ) Table shows the tumorigenicity of different subsets of breast cancer cells isolated on the basis of CD44, CD44 expression levels compared with the induced subset. Tumorigenicity was quantified by implanting different cell numbers in mice and monitoring the number of tumours developed. ( c ) Graph shows the temporal kinetics of tumour growth initiated from seeding CD44 Hi , CD44 Lo or induced cells. ( d ) Schematic illustrates experimental design of in vivo kinetic analysis of tumour growth of distinct subpopulations. Cells were isolated from each quadrant and an equal number of cells were implanted in mice in separate areas. An induced CD44 Hi CD24 Hi group was run in parallel. ( e ) Graph shows tumour volumes from indicated phenotypic subsets over time. Data shown are mean±s.e.m., n =4.

Article Snippet: Following primary antibodies were used: anti human Ki-67 (rabbit polyclonal from Vector Laboratory, 1:600 dilution), anti-human cleaved caspase3 (rabbit polyclonal, clone D175, Cell Signaling Technology, Cambridge, MA, USA), Anti-human CD44 (Clone IM7), P-Hck Y410 (Cell Signaling Technology).

Techniques: Selection, Isolation, Expressing, In Vivo

Journal: Nature Communications

Article Title: Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition

doi: 10.1038/ncomms7139

Figure Lengend Snippet: ( a ) Graph indicates tumour volume over time from heterotopic, syngeneic murine mammary carcinoma model (4T1). Groups were treated with docetaxel (DTX) or vehicle (black arrows show treatment days). Tumours were extracted on day 9 and 19 (Red arrows) corresponding to plateau or regrowth of tumour volume in docetaxel-treated arms, respectively (Data shown are mean±s.e.m., n =4). ( b ) Representative IHC of CD44 and pHCK following tumour extraction at indicated time points, H&E from serial sections confirm viable regions of tumour. Scale bar, 50 μm ( c ) Schematic shows experimental design for temporal delivery of dasatinib (10 mg kg −1 ) administered in two schedules, (1) 72 h or (2) 216 h post DTX treatment. The first schedule is designed to target the induction phase of chemotherapy-phenotypic transitioning and the second schedule targets the recalibration phase to parental state. ( d ) Histogram quantifies specific tumour growth rate. ( e ) Kaplan–Meier survival graph of an orthotopic syngeneic mammary carcinoma model treated as indicated ( N =4 in all groups, N =3 for vehicle). ( f ) Representative confocal microscopy shows co-staining of CD44 and pHck. Regions of robust Hck activity correspond to regions of tumour with high expression of CD44 (areas outside yellow circumscription). Scale bar, 25 μm

Article Snippet: Following primary antibodies were used: anti human Ki-67 (rabbit polyclonal from Vector Laboratory, 1:600 dilution), anti-human cleaved caspase3 (rabbit polyclonal, clone D175, Cell Signaling Technology, Cambridge, MA, USA), Anti-human CD44 (Clone IM7), P-Hck Y410 (Cell Signaling Technology).

Techniques: Extraction, Confocal Microscopy, Staining, Activity Assay, Expressing

Journal: Nature Communications

Article Title: Temporally sequenced anticancer drugs overcome adaptive resistance by targeting a vulnerable chemotherapy-induced phenotypic transition

doi: 10.1038/ncomms7139

Figure Lengend Snippet: ( a ) Representative H&E and IHC of activated caspase-3 in human taxane-refractory tumour explant treated with docetaxel or a combination of docetaxel and dasatinib. Histogram shows IHC score quantification of activated caspase-3 ( N =6, ** P <0.01). Data shown are mean±s.e.m. from independent replicates. Scale bar, 100 μm ( b ) Schematic shows the treatment of parent drug-naive cells with cytotoxic chemotherapy (taxane) confers phenotypic plasticity transitioning the population towards a transient drug-tolerant state, arising through clustering of CD44 and CD24 in lipid rafts, HCK activation and suppression of proapoptotic signalling via nuclear translocation. This transient state is vulnerable to inhibition of SFK using kinase inhibitors, leading to apoptosis. The same SFK inhibitors have no effect on parent cells.

Article Snippet: Following primary antibodies were used: anti human Ki-67 (rabbit polyclonal from Vector Laboratory, 1:600 dilution), anti-human cleaved caspase3 (rabbit polyclonal, clone D175, Cell Signaling Technology, Cambridge, MA, USA), Anti-human CD44 (Clone IM7), P-Hck Y410 (Cell Signaling Technology).

Techniques: Activation Assay, Translocation Assay, Inhibition