Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: Livers were collected from mice (n=6) sacrificed at respective time-points (2,4,8,12,24 & 48h for ANIT; 24 & 48h for CCl4; 6 & 24h for AlOH; and 10 d for BDL). RNA was isolated and cDNA was made using a commercial MMLV-RT kit. The cDNA samples were analyzed by quantitative RT-PCR using Fmo3 mouse-specific primers. Gene expression was normalized to the housekeeping gene β-actin. Fmo3 mRNA expression is presented as mean Fold Change ± SE. One-way ANOVA, t-test or two-way ANOVA was performed, appropriately, followed by the Dunnett's posttest for One-way ANOVA and the Bonferroni posttest for two-way ANOVA. Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated and hepatotoxicant-treated or BDL group.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Isolation, Quantitative RT-PCR, Expressing

Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: Western immunoblots for Fmo3 were performed using liver microsomes from control and hepatotoxicant-treated or BDL mice. A custom-made rabbit anti-mouse Fmo3 primary antibody, described in Materials and Methods was used to detect Fmo3. Fmo3 protein levels were normalized to β-actin loading control. Microsomal proteins isolated from naïve female mouse liver were used as a positive control (indicated by “+” sign). The data are presented as blots and as mean Fmo3 protein expression (Fold Change) ± SE. One-way ANOVA, t-test or two-way ANOVA was performed, appropriately, followed by the Dunnett's posttest for One-way ANOVA and the Bonferroni posttest for two-way ANOVA. Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated and hepatotoxicant-treated or BDL group.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Western Blot, Isolation, Positive Control, Expressing

Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: Plasma and livers were collected from mice 72 h following APAP (400 mg/kg) or vehicle treatment. (A) The data are presented as mean plasma ALT (IU/L) ± SE. (B) RNA was isolated from livers andcDNA samples were analyzed by quantitative RT-PCR using Fmo3 mouse-specific primers. Gene expression was normalized to the housekeeping gene β-actin. Fmo3 mRNA expression are presented as mean Fold Change ± SE. Oneway ANOVA was performed followed by the Dunnett's post-test. Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated group and APAP-treated group and hash (#) represent a statistical difference (p<0.05) compared with APAP-treated wild-type mice.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Isolation, Quantitative RT-PCR, Expressing

Journal: Toxicology

Article Title: Differential Fmo3 Gene Expression in Various Liver Injury Models Involving Hepatic Oxidative Stress in Mice

doi: 10.1016/j.tox.2014.08.013

Figure Lengend Snippet: After overnight fasting, groups of wild-type and Nrf2 knockout mice received a single dose of 400 mg/kg APAP or vehicle. Livers were collected 72 h following APAP or vehicle treatments. Western blot for Fmo3 was performed using liver microsomes from control and APAP-treated mice. Equal protein loading (10 μg protein/lane) was confirmed by detection of β-actin. Microsomal proteins isolated from naïve female mouse liver were used as a positive control indicated by “+” sign. The data are presented as blots and as mean Fmo3 protein expression (Fold Change) ± SE (A). FMO activity was measured in liver microsomes from control and APAP-treated mice using methimazole as substrate. Data are presented as mean Specific Activity (μM/min/mg) ± SE (B). Asterisks (*) represent a statistical difference (p < 0.05) between vehicle-treated group and APAP-treated group and hash (#) represent a statistical difference (p<0.05) compared with APAP-treated wild-type mice.

Article Snippet: Membranes were blocked with 5 % non-fat powdered milk in tris buffered saline containing 0.05% tween-20(TBS-T) for 8 h. A rabbit anti-mouse Fmo3 primary antibody (GenScript USA Inc., NJ) (1:5000) was used to detect Fmo3 with β-actin as a loading control.

Techniques: Knock-Out, Western Blot, Isolation, Positive Control, Expressing, Activity Assay

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) Sequence logos for ER resident proteins with C-terminal KDEL retrieval signals and variants thereof calculated using frequency or protein abundance ( ; ). ( b ) Combined cellular concentrations of ER resident proteins with canonical KDEL, RDEL, and HDEL retrieval sequences in HeLa cells and mouse brain. ( c ) Competition binding assays for [ 3 H]-TAEHDEL and unlabelled TAEKDEL, TAERDEL, and TAEHDEL to the KDEL receptor. IC 50 values for the competing peptides were used to calculate the apparent K D with the Cheng-Prusoff equation . ( d ) Endogenous KDEL receptor redistribution was measured in COS-7 cells in the absence (-ligand) or presence of K/R/H/A/DDEL (mScarlet-xDEL sec ). TGN46 was used as a Golgi marker. Scale bar is 10 µm. ( e ) The mean difference for K/R/H/A/DDEL comparisons against the shared no ligand control are shown as Cummings estimation plots. The individual data points for the fraction of KDEL receptor fluorescence in the Golgi are plotted on the upper axes with sample sizes and p values. Figure 1—source data 1. Source data for the ligand-induced KDELR receptor retrieval assays in .

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Sequencing, Binding Assay, Marker, Fluorescence

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) Crystal structure of chicken KDELR2 viewed from the side with the transmembrane helices numbered and coloured from N-terminus (blue) to C-terminus (red). The predicted membrane-embedded region of the receptor is indicated by a grey shaded box, with labels at the luminal and cytoplasmic faces. The TAEHDEL peptide is shown in stick format, coloured grey. ( b ) Close up views of bound TAEHDEL (this study), ( c ) TAERDEL (this study), and ( d ) TAEKDEL (PDB:6I6H) peptides bound to the receptor are shown with contributing side chains labelled. Hydrogen bonds are indicated as dashed lines. The molecular orbitals of W120 and the −4 histidine on the peptide are shown as a dotted surface. ( e ) Superposition of the HDEL, RDEL, and KDEL peptides reveals near identical binding position within the receptor. Retrieval signal side chains are numbered counting down from the C-terminus.

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Binding Assay

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) Normalised binding of [ 3 H]-TAEKDEL and ( b ) [ 3 H]-TAEHDEL signals to purified WT and the indicated E117 and W120 mutant variants of chicken KDELR2. Bar graphs show mean binding ± SEM (n = 3). Line graphs show titration binding assays. ( c ) The fraction of WT, E117, and W120 mutant KDEL receptor localised to the Golgi in COS-7 cells was measured before (no ligand) and after challenge with different retrieval signals (K/R/HDEL) as indicated. Effect sizes are shown as the mean difference for K/R/HDEL comparisons against the shared -ligand control with sample sizes and p-values. Also see files. ( d ) The π-π interactions between W120 and the histidine were visualised using reduced density gradient analysis. The wild-type W120 exhibit stronger π-π interactions compared with W120F, while W120A shows no π-π interactions. ( e ) When W120 is changed to phenylalanine, the protonated histidine has a higher root mean squared fluctuation (RMSF) in the binding pocket, which is further increased for the W120A substitution. ( f ) Binding of [ 3 H]-TAEHDEL to the KDEL receptor was measured at pH 5.4–7.0 and is plotted as a function of receptor concentration.

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Binding Assay, Purification, Mutagenesis, Titration, Concentration Assay

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) E117Q, E117N, or E117A mutant KDEL receptors were tested for K/A/DDEL-induced redistribution from Golgi to ER in COS-7 cells. KDEL receptor distribution was followed in the absence (-ligand) or presence of K/A/DDEL sec . TGN46 was used as a Golgi marker. Scale bar is 10 µm. ( b ) The fraction of E117Q, E117N or E117A mutant KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with different retrieval signals (K/A/DDEL). Effect sizes are shown as the mean difference for K/A/DDEL comparisons against the shared -ligand control with sample sizes and p values. . Figure 4—source data 1. Source data for the ligand-induced KDELR receptor retrieval assays in .

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Mutagenesis, Marker

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) KDEL receptor sequence alignment showing two regions centred around amino acid D50 and W120 of the human proteins. Cognate retrieval signal variants are shown to the right of the alignment. ( b ) The structure of the KDEL receptor with bound TAEHDEL highlighting key residues involved in ligand binding and variant residues D50, N54, and E117. ( c ) The charged surface for the WT KDEL receptor and ( d ) N50, N50/K54 and N50/K54/Q117 mutants is shown.

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Sequencing, Ligand Binding Assay, Variant Assay

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) WT and a series of ‘K. lacti s’-like mutant KDEL receptors were tested for K/A/DDEL-induced redistribution from Golgi to ER in COS-7 cells. KDEL receptor distribution was followed in the absence (-ligand) or presence of K/A/DDEL sec . TGN46 was used as a Golgi marker. Scale bar is 10 µm. ( b ) The fraction of WT and mutant KDEL receptor localised to the Golgi was measured before (no ligand) after challenge with different retrieval signals (K/A/DDEL). Effect sizes are shown as the mean difference for K/A/DDEL comparisons against the shared -ligand control with sample sizes and p values. Figure 6—source data 1. Source data for the ligand-induced KDELR receptor retrieval assays in .

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Mutagenesis, Marker

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet: ( a ) Images depicting the key stages (i.-iv.) of TAEKDEL binding to the wild-type (WT) KDEL receptor simulated using molecular dynamics. Initial engagement of the C-terminus to R169 (i) is followed by transfer to R5 (ii), shortly followed by interaction of E −2 with R169 (iii). Finally, R47 engages the C-terminus allowing D −3 to interact with R169 (iv). See also . ( b ) A carton model depicting the key stages of retrieval signal binding and final pH-dependent locked state. ( c ) Occupancy of the hydrogen bonds between the C-terminus of the KDEL retrieval signal and R5, R47, and R169 is plotted as a function of signal position within the binding pocket. ( d ) The occupancy of potential hydrogen bonds between the different positions of the KDEL retrieval signal and D50, S54, and E117 is plotted as a function of signal position within the binding pocket. ( e ) Competition binding assays for [ 3 H]-TAEHDEL and unlabelled TAEKDEL and TAEHDEL with a free (COOH) or amidated (CONH) C-terminus to chicken KDELR2 showing IC 50 values for the competing peptides. ( f ) Normalised binding of [ 3 H]-TAEHDEL and [ 3 H]-TAEKDEL signals to the purified WT H12A, R169A, or R169K mutant chicken KDELR2. A mock binding control with no receptor indicates the background signal. ( g ) Distribution of WT, H12A, R169A, and R169K KDEL receptors was measured in COS-7 cells in the absence (-ligand) or presence of K/R/HDEL sec . The mean differences for K/R/HDEL comparisons against the shared no ligand control are shown with sample sizes and p values. See also with accompanying source data.

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Binding Assay, Purification, Mutagenesis

Journal: eLife

Article Title: A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

doi: 10.7554/eLife.68380

Figure Lengend Snippet:

Article Snippet: All samples were analysed by Western blotting (Trans-Blot Turbo transfer system, Bio-Rad) for xDEL ligand (mouse anti-RFP 6G6, Chromotek), resident ER chaperones BIP (rabbit #ab21685, Abcam), PDI (rabbit #11245–1, ProteinTech), ERP72 (rabbit #5033S, Cell Signalling Technology), ERP44 (rabbit #3798S, Cell Signalling Technology) and the KDEL receptor (mouse ADI-VAA-PT048, Enzo Life Sciences).

Techniques: Plasmid Preparation, Expressing, Western Blot, Recombinant, Functional Assay, Variant Assay, Software, Microscopy, Modification

Journal: Nanoscale Research Letters

Article Title: A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells

doi: 10.1186/s11671-015-0972-6

Figure Lengend Snippet: Fluorescence microscopy images of hNSCs cultured with PCL conduit for 14 days: a DAPI and b β-III tubulin. DAPI staining is indicated in blue and β-III tubulin staining is indicated in green. Arrow indicates the direction of aligned nanofibers

Article Snippet: After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C.

Techniques: Fluorescence, Microscopy, Cell Culture, Staining

Journal: Nanoscale Research Letters

Article Title: A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells

doi: 10.1186/s11671-015-0972-6

Figure Lengend Snippet: Confocal microscopy images of hNSCs cultured with PSHU-RGD/PCL conduit for 14 days: representative images a , b along the center and e , f inner wall of microchannels, a , d DAPI, b , e β-III tubulin, and c , f combined DAPI and β-III tubulin images. DAPI staining is indicated in blue and β-III tubulin staining is indicated in green . Images show that hNSCs migrated far into the conduit with extensive neurite extension along the nanofiber direction ( arrows )

Article Snippet: After cryosectioning (20-μm slices), each section was washed twice in PBS for 10 min. Then, the samples were treated with blocking buffer (3 % goat serum PBS with 0.01 % Triton X-100) for 1 h at room temperature before adding β-III tubulin goat anti-mouse primary antibody (1:200 dilution, Promega, Madison, WI, USA) overnight at 4 °C.

Techniques: Confocal Microscopy, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: List of mAb used for the analysis of lymphocyte subsets. BD (BD Biosciences, San Jose, CA); eB (eBioscience, San Diego, CA); DN: double negative; DP: double positive; SP: single positive; T CM : central memory T cell; T EM : effector memory T cell; CD26 expression was assessed in all lymphocyte subsets.

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Expressing, Marker

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: Splenocytes were obtained at 4 and 6 weeks after treatment. A) Percentage of T effector memory (EM) , T central memory (CM) and naïve T lymphocytes (CD8 + and CD4 + ) in control (white circles) and treated mice (black circles). B) Representative FACS plots of the naïve (CD3 + CD4 + /CD8 + CD44 low CD62L + ), T CM (CD3 + CD4 + /CD8 + CD44 high CD62L + ) and T EM (CD3 + CD4 + /CD8 + CD44 high CD62L neg ) both for CD4 + and CD8 + T lymphocytes from spleen in treated and control mice at 4 weeks of treatment. C) MFI for CD26 expression on T EM, T CM and naïve T lymphocytes (CD8 + and CD4 + ) in control (white circles) and treated mice (black circles). Lines represent the mean of 8–10 mice. Comparison between treated and untreated mice showed an increased expression of CD26 on CD8 + T EM cells at any of the study’s time-points. (*p<0.05, **p<0.01, three way Anova).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, Expressing, Comparison

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: T cells from PLNs were obtained from control (white circles) and treated (black circles) NOD mice at 4 weeks after treatment. A) Percentage of T effector memory (EM) , T central memory (CM) and naïve T lymphocytes (CD8 + and CD4 + ). B) MFI for CD26 expression on T EM, T CM and naïve T lymphocytes (CD8 + and CD4 + ). The expression of CD26 was significantly increased on CD4 + T EM and CD8 + T EM cells in MK626-treated mice when compared to controls. Lines represent the mean of 6 mice (*p<0.05, ***p<0.001, three way Anova).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, Expressing

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: A) Percentage of CD8 + and CD4 + T lymphocytes infiltrating pancreatic islets in untreated (white circles) and treated (black circles) mice assessed by flow cytometry. Lines represent the mean of 6–9 mice. No statistical differences were observed between groups (Mann-Whitney test). B) Expression of CD26 determined by flow cytometry (MFI) in CD8 + and CD4 + T lymphocytes from control (white circles) and treated (black circles) mice. Lines represent the mean of 4–8 mice. (*p≤0.05, Mann-Whitney test).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Flow Cytometry, MANN-WHITNEY, Expressing, Control

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: CD8 + T EM cell from control (n = 9) and treated (n = 9) mice obtained at 4 weeks of treatment were stimulated with anti–CD3 in vitro . A) Proliferative rate in CD8 + T EM lymphocytes from MK626-treated mice (black circles) and in control group (white circles). T cell proliferation was expressed as counts per minute (c.p.m) using 3 H-TdR. B) Cytokine concentration (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α and TGF-β) in supernatant from proliferation assays in treated mice (black bars) compared to control mice (white bars). Results are expressed as mean ± SEM. Mann-Whitney test was used for the evaluation of statistical significance (*p<0.05;**p<0.01).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, In Vitro, Concentration Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: A) Representative dot plots showing CFSE-T lymphocytes, stimulated with insulin-loaded mDCs, in the presence or absence of CTV-CD8 + T EM lymphocytes (ratio 1:1) and MK626 (10μM). B) Percentage of proliferating CFSE low T lymphocytes versus all T lymphocytes, with insulin-loaded mDCs (circles and triangles), in the presence of CTV-CD8 + T EM lymphocytes (triangles) and MK626 (open symbols) for 3 days. Plots show the mean (line) of three independent experiments.

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques:

Journal: The FASEB Journal

Article Title: Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality

doi: 10.1096/fj.201900246RR

Figure Lengend Snippet: Genetic deletion of Per1/Per2 enhances pulmonary leukocyte infiltration and lung tissue damage, which contribute to an early mortality in SCD. A) Schematic of SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. B) Images of irradiated male mice ∼16 wk post-BMT. C) Increased mortality was identified beginning 42 d and up to 112 d after irradiation and hematopoietic adoptive transfer in female SCD BM transplanted to Per1/Per2 dKO mice. D) Panel of histologic stains, hematoxylin and eosin (H&E), and Ly6-positive leukocytes as indicated with red arrows in SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. E) Semiquantification of histologic assessment of lung isolated from SCD or WT phenotypic mice with or without Per1/Per2. F) Quantification of leukocytes in ×20 field in whole lung sections isolated from SCD and WT BM transplant mice; n = 5 mice/group. ND, not determined. *P < 0.05, SCD → WT compared with WT → WT mice, **P < 0.01, SCD → WT compared with SCD → Per1/Per2 dKO mice.

Article Snippet: Sections were incubated with Ly6 primary antibody (rat monoclonal anti-mouse, 1:500 dilution in blocking solution; BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Irradiation, Adoptive Transfer Assay, Isolation