Journal: Animal Biotechnology

Article Title: Hypoxic stress caused apoptosis of MDBK cells by p53/BCL6-mitochondrial apoptosis pathway

doi: 10.1080/10495398.2023.2299241

Figure Lengend Snippet: Hypoxic stress promotes apoptosis of MDBK cells through mitochondrial apoptotic pathway. A: Apoptosis rate of MDBK cells under hypoxic environment; B: subcellular localization of Cyt c in MDBK cells treated with hypoxic environment; C–D: the expression of Caspase-9 and Caspase-3 in MDBK cells treated with hypoxic environment. In , the apoptosis rate of MDBK cells in normal culture group (N) was set to ‘1’. in , the expression of Caspase-9, Caspase-3 and BCL6 of MDBK cells in normal culture group (N) was set to ‘1’. N: MDBK cells cultured in standard environment; H: MDBK cells cultured in hypoxic environmentin. In the bar charts, different superscript lowercase letters indicated significant differences ( p < 0.05 ), while the same letters represent no significant difference ( p > 0.05 ).

Article Snippet: Primary antibodies used in this study were as follows: BCL6 antibody (1:500, bsm-60223R, Bioss, Beijing, China); p53 antibody (1:1,000, ab26, Abcam, Cambridgeshire, UK); β-actin antibody (1:500, bsm-33036M, Bioss); Cytc Antibody (1:500, bs-0013R, Bioss); β-Tubulin antibody (1:1,000, ab179511, Abcam), VDAC1 Antibody (1:1,000, ab15895, Abcam); Caspase-3 antibody (1:500, bs-33199M, Bioss); Caspase-9 (1:500, bs-0049R, Bioss).

Techniques: Expressing, Cell Culture

Journal: Animal Biotechnology

Article Title: Hypoxic stress caused apoptosis of MDBK cells by p53/BCL6-mitochondrial apoptosis pathway

doi: 10.1080/10495398.2023.2299241

Figure Lengend Snippet: Hypoxic stress promotes apoptosis of MDBK cells through BCL6. A: Apoptotic rate of MDBK cells treated with hypoxic environment or BCL6 gene overexpression; B-C: the expression of Caspase-9 and Caspase-3 in MDBK cells treated with hypoxic environment or BCL6 gene overexpression. D: the apoptosis rate of MDBK cells treated with hypoxic environment or BCL6 gene silencing; E-F: the expression of Caspase-9 and Caspase-3 in MDBK cells treated with hypoxic environment or BCL6 gene silencing. In and D, the apoptosis rate of MDBK cells in normal culture group (N) was set to ‘1’. in and F, the expression of Caspase-9 and Caspase-3 of MDBK cells in normal culture group (N) was set to ‘1’. N: MDBK cells cultured in standard environment; H: MDBK cells cultured in hypoxic environment. EV: cells were transfected with empty vector. NC: cells were transfected with negative control siRNA. BCL6 GO: cells were transfected with BCL6 gene overexpression vector. si-BCL6: cells were transfected with BCL6 gene siRNA. In the bar charts, different superscript lowercase letters indicate significant differences ( p < 0.05 ), while the same letters represent no significant difference ( p > 0.05 ).

Article Snippet: Primary antibodies used in this study were as follows: BCL6 antibody (1:500, bsm-60223R, Bioss, Beijing, China); p53 antibody (1:1,000, ab26, Abcam, Cambridgeshire, UK); β-actin antibody (1:500, bsm-33036M, Bioss); Cytc Antibody (1:500, bs-0013R, Bioss); β-Tubulin antibody (1:1,000, ab179511, Abcam), VDAC1 Antibody (1:1,000, ab15895, Abcam); Caspase-3 antibody (1:500, bs-33199M, Bioss); Caspase-9 (1:500, bs-0049R, Bioss).

Techniques: Over Expression, Expressing, Cell Culture, Transfection, Plasmid Preparation, Negative Control

Journal: Animal Biotechnology

Article Title: Hypoxic stress caused apoptosis of MDBK cells by p53/BCL6-mitochondrial apoptosis pathway

doi: 10.1080/10495398.2023.2299241

Figure Lengend Snippet: BCL6 negatively regulates apoptosis of MDBK cells. A: the apoptosis rate of MDBK cells treated with BCL6 gene overexpression; B-C: the expression of Caspase-9 and Caspase-3 in MDBK cells treated with BCL6 gene overexpression. D: the apoptosis rate of MDBK cells treated with BCL6 gene silencing; E-F: the expression of Caspase-9 and Caspase-3 in MDBK cells treated with BCL6 gene silencing. In and D, the apoptosis rate of MDBK cells with no transfected group (B) was set to ‘1’. in and F, the expression of Caspase-9 and Caspase-3 of MDBK cells with no transfected group (B) was set to ‘1’. B: Cells were no transfected; EV: cells were transfected with empty vector. NC: cells were transfected with negative control siRNA. BCL6 GO: cells were transfected with BCL6 gene overexpression vector. si-BCL6: cells were transfected with BCL6 gene siRNA. In the bar charts, different superscript lowercase letters indicate significant differences ( p < 0.05 ), while the same letters represent no significant difference ( p > 0.05 ).

Article Snippet: Primary antibodies used in this study were as follows: BCL6 antibody (1:500, bsm-60223R, Bioss, Beijing, China); p53 antibody (1:1,000, ab26, Abcam, Cambridgeshire, UK); β-actin antibody (1:500, bsm-33036M, Bioss); Cytc Antibody (1:500, bs-0013R, Bioss); β-Tubulin antibody (1:1,000, ab179511, Abcam), VDAC1 Antibody (1:1,000, ab15895, Abcam); Caspase-3 antibody (1:500, bs-33199M, Bioss); Caspase-9 (1:500, bs-0049R, Bioss).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Negative Control

Journal: Animal Biotechnology

Article Title: Hypoxic stress caused apoptosis of MDBK cells by p53/BCL6-mitochondrial apoptosis pathway

doi: 10.1080/10495398.2023.2299241

Figure Lengend Snippet: Hypoxic stress negatively regulates the expression of BCL6 via p53 in MDBK cells. A-B: the expression of BCL6 in MDBK cells treated with hypoxic environment or p53 gene overexpression; C-D: the expression of BCL6 in MDBK cells treated with hypoxic environment or p53 gene silencing. In , the expression of BCL6 of MDBK cells in normal culture group (N) was set to ‘1’. N: MDBK cells cultured in standard environment; H: MDBK cells cultured in hypoxic environment. EV: cells were transfected with empty vector. NC: cells were transfected with negative control siRNA. p53 GO: cells were transfected with p53 gene overexpression vector. si-p53: cells were transfected with p53 gene siRNA. In the bar charts, different superscript lowercase letters indicate significant differences ( p < 0.05 ), while the same letters represent no significant difference ( p > 0.05 ).

Article Snippet: Primary antibodies used in this study were as follows: BCL6 antibody (1:500, bsm-60223R, Bioss, Beijing, China); p53 antibody (1:1,000, ab26, Abcam, Cambridgeshire, UK); β-actin antibody (1:500, bsm-33036M, Bioss); Cytc Antibody (1:500, bs-0013R, Bioss); β-Tubulin antibody (1:1,000, ab179511, Abcam), VDAC1 Antibody (1:1,000, ab15895, Abcam); Caspase-3 antibody (1:500, bs-33199M, Bioss); Caspase-9 (1:500, bs-0049R, Bioss).

Techniques: Expressing, Over Expression, Cell Culture, Transfection, Plasmid Preparation, Negative Control

Journal: Oncology Letters

Article Title: Immunohistochemical profile and prognostic significance in primary central nervous system lymphoma: Analysis of 89 cases

doi: 10.3892/ol.2017.6893

Figure Lengend Snippet: Immunohistochemical labeling. (A and B) H&E staining. In tumor cells with the diffuse distribution, the size of nuclei were 2 times greater than of normal lymphocytes. (A) Magnification, ×200. (B) Magnification, ×400. (C) CD20 cell membrane staining performed using the EnVision method. Magnification, ×200. (D) CD10 cell membrane staining performed using the EnVision method. Magnification, ×200. (E) BCL-6, nuclei staining, EnVision method, ×100. (F) MUM-1 nuclei staining performed using the EnVision method. Magnification, ×200. (G) BCL-2 cytoplasmic staining performed using the EnVision method. Magnification, ×200. (H) Ki-67 nuclei staining performed using the EnVision method. Magnification, ×200. H&E, hematoxylin and eosin; CD, cluster of differentiation; BCL, B cell lymphoma; MUM-1, multiple myeloma-1.

Article Snippet: Monoclonal antibodies against CD20 (UM800002, 1:100 dilution), CD10 (UM870127, 1:600 dilution), BCL-6 (TA804186, 1:150 dilution), MUM1 (TA327705; 1:100-1:500 dilution), BCL-2 (UM870117, 1:500 dilution), Ki-67 (TA352729, 1:100), CD138 (TA327619, 1:25-1:200) were used and all purchased from OriGene Technologies, Inc. (Rockville, MD, USA).

Techniques: Immunohistochemical staining, Labeling, Staining

Journal: Nature immunology

Article Title: Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions

doi: 10.1038/s41590-020-0781-7

Figure Lengend Snippet: 20,000-100,000 naive LLO56 and LLO118 cells were co-transferred into recipient B6 mice and then infected with actA-Lm the following day. Spleens were harvested on the indicated days post-infection for flow cytometry analysis of the activated LLO T cell populations . Data collected from each individual recipient mouse are paired. a , Representative flow plots depicting Teff, pre-Tfh, and Tfh PD-1/CXCR5 gating strategies. Numbers shown are the frequency of each subset within the activated LLO parent population. b , Quantification of the frequency of Tfh cells. Three independent experiments for day 4 (n=13), eight for day 7 (n=31), and two for day 10 (n=10). c , Total numbers of activated LLO T cells. Three independent experiments for days 4 (n=13) and 7 (n=14), two for day 10 (n=10). d , Total numbers of LLO Tfh cells from the same experiments as in (c). e , Percentage difference in Bcl6 MFI of the paired pre-Tfh and Tfh subsets for each genotype relative to the average B6 Teff subset at day 7 post-infection. Data are from the same experiments as in (c) and exclude mice with no LLO56 Tfh generation. f , The frequency of Teff and pre-Tfh cells from the same experiments as in (b). g , LLO T cells were assessed for Tbet, GATA-3, and RORγt expression via intracellular staining at day 7 post-infection. Three independent experiments (n=14) are shown. h , Splenocytes were stimulated with PMA and ionomycin before intracellular cytokine staining was performed to assess frequency and i , MFI of IFN-γ expression in the activated LLO populations. Three independent experiments for days 4 (n=15) and 7 (n=14) and two for day 10 (n=7). MFI data shows the mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 . Paired t test or Wilcoxon matched-pairs signed rank test for nonnormally distributed data (b-d, f-h). Two-way ANOVA using tukey’s multiple comparisons test for comparison of subsets across genotypes and Sidak’s multiple comparisons test for comparisons among subsets within each genotype (e). One-way ANOVA analysis (i).

Article Snippet: The following antibodies were used for intracellular flow cytometry analysis: anti-Bcl6 (PE; clone BCL-DWN; Fisher Scientific; cat. no. 501122326; 1:100 dilution), anti-Tbet (PE; clone eBio4B10; Fisher Scientific; cat. no. 5010893), anti-GATA-3 (PE-Cy7; clone TWAJ; Fisher Scientific; cat. no. 501129305), anti-RORγt (BV421; clone Q31-378; BD Biosciences; cat. no. 562894), anti-IFN-γ (FITC; clone XMG1.2; Biolegend; cat. no. 505806), anti-IL-4 (Biotin; clone BVD6-24G2; BD Biosciences; cat. no. 554390; 1:100 dilution), anti-IL-21 (PE; clone FFA21; Fisher Scientific; cat. no. 5011172; 1:100 dilution), anti-H2-DM (clone 2E5a; Fisher Scientific; cat. no. 552405), anti-rat IgG1 (FITC; clone RG11/39.4; Fisher Scientific; cat. no. BDB553892), and anti-Nur77 (PE; clone 12.14; Fisher Scientific; cat. no. 5011028).

Techniques: Infection, Flow Cytometry, Expressing, Staining, Comparison

Journal: Nature immunology

Article Title: Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions

doi: 10.1038/s41590-020-0781-7

Figure Lengend Snippet: 100,000 naive CD4 + T cells of each LLO genotype were co-transferred into recipient B6 mice and infected with actA-Lm the following day. Spleens were harvested post-infection for flow cytometry. a , Frequencies of ICOS + cells and b , CD40L + cells in the Teff, pre-Tfh, and Tfh subsets of the LLO populations. Data points from individual recipient mice are paired. Three independent experiments for both days 4 (n=15) and 7 (n=14). c , On the indicated day post-infection, splenocytes were harvested and stimulated with PMA and ionomycin before intracellular cytokine staining was performed. The frequency of cytokine producing cells as well as cytokine MFI, given as a ratio of LLO118/LLO56 for each recipient mouse, are shown for IL-4 and d , IL-21. For both (c) and (d), three independent experiments were performed for day 4 (n=14) and two for days 7 (n=10) and 10 (n=7). MFI data show the mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 . Two-way ANOVA or tukey’s multiple comparisons test (a). Paired t test or Wilcoxon matched-pairs signed rank test for nonnormally distributed data (b-d). One-way ANOVA analysis (MFI data for c, d).

Article Snippet: The following antibodies were used for intracellular flow cytometry analysis: anti-Bcl6 (PE; clone BCL-DWN; Fisher Scientific; cat. no. 501122326; 1:100 dilution), anti-Tbet (PE; clone eBio4B10; Fisher Scientific; cat. no. 5010893), anti-GATA-3 (PE-Cy7; clone TWAJ; Fisher Scientific; cat. no. 501129305), anti-RORγt (BV421; clone Q31-378; BD Biosciences; cat. no. 562894), anti-IFN-γ (FITC; clone XMG1.2; Biolegend; cat. no. 505806), anti-IL-4 (Biotin; clone BVD6-24G2; BD Biosciences; cat. no. 554390; 1:100 dilution), anti-IL-21 (PE; clone FFA21; Fisher Scientific; cat. no. 5011172; 1:100 dilution), anti-H2-DM (clone 2E5a; Fisher Scientific; cat. no. 552405), anti-rat IgG1 (FITC; clone RG11/39.4; Fisher Scientific; cat. no. BDB553892), and anti-Nur77 (PE; clone 12.14; Fisher Scientific; cat. no. 5011028).

Techniques: Infection, Flow Cytometry, Staining

Journal: Nature immunology

Article Title: Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions

doi: 10.1038/s41590-020-0781-7

Figure Lengend Snippet: 3,000 LLO56 or LLO118 cells were transferred into recipient Tcra −/− mice and immunized one day later with NP-LLO LT -N. On day 7 post-immunization, splenocytes were analyzed by flow cytometry for a , total number of activated LLO T cells, b , Tfh cell frequency of the LLO T cell populations, c , total number of LLO Tfh cells, and d , frequency of GC, NP + -B cells (see for gating) Six independent experiments (n=16 for LLO56 and n=14 for LLO118). e , 3000 LLO56 or LLO118 cells were transferred and activated as in (a). Spleens were harvested post-immunization for immunohistochemistry analysis. GC staining: PNA (yellow), IgD (white), and Hoechst (blue). Representative images are shown for three independent experiments at day 7 (n=6 for LLO56 and LLO118, n=4 for controls) and two at d10 (n=3 for LLO56 and LLO118, n=4 for controls). Scale bars (bottom left corners) = 300μm. Controls are Tcra −/− mice that were immunized with NP-LLO LT -N but had received no prior T cell transfers (representative images in ). f , For images obtained in (e), quantification of the number of GCs is shown. GCs were manually counted in a blinded manner and then normalized to spleen volume. Two sections per individual mouse were averaged. g , Tcra −/− mice receiving either 3,000 (data represented by the left Y axis) or 20,000 (data represented by the right Y axis) LLO56 or LLO118 cells were immunized with NP-LLO LT -N, and serum was collected on days 14, 21, and 100 post-immunization. Endpoint titers were determined with serum from Tcra −/− mice that had received LLO T cell transfers but were immunized with unconjugated protein, LLO LT -N. High- and low-affinity antibodies were determined by coating ELISA plates with NP(2)-BSA and NP(28)-BSA, respectively. For 3,000 cell transfers, data represent three independent experiments for all time-points [(d14: n=8 for LLO56, n=7 for LLO118), (d21: n=11 for LLO56, n=9 for LLO118), (d100: n=10 for both LLO genotypes)]. For 20,000 cell transfers, four independent experiments for day 14 (n=16 for LLO56, n=19 for LLO118), three for d21 (n=13 for both LLO genotypes), and two for day 100 (n=8 for both LLO genotypes). h , 20,000 LLO56 or LLO118 cells were transferred and activated as in (a). On day 7 post-immunization, splenocytes were analyzed by flow cytometry for the total number of activated LLO T cells, i , frequency of Tfh cells within the LLO populations, j , total number of LLO Tfh cells, and k , frequency of NP + GC B cells. Three independent experiments (n=9). All data represent mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 . Unpaired t test or Mann-Whitney test for nonnormally distributed data (a-d, g-k). One-way ANOVA (f).

Article Snippet: The following antibodies were used for intracellular flow cytometry analysis: anti-Bcl6 (PE; clone BCL-DWN; Fisher Scientific; cat. no. 501122326; 1:100 dilution), anti-Tbet (PE; clone eBio4B10; Fisher Scientific; cat. no. 5010893), anti-GATA-3 (PE-Cy7; clone TWAJ; Fisher Scientific; cat. no. 501129305), anti-RORγt (BV421; clone Q31-378; BD Biosciences; cat. no. 562894), anti-IFN-γ (FITC; clone XMG1.2; Biolegend; cat. no. 505806), anti-IL-4 (Biotin; clone BVD6-24G2; BD Biosciences; cat. no. 554390; 1:100 dilution), anti-IL-21 (PE; clone FFA21; Fisher Scientific; cat. no. 5011172; 1:100 dilution), anti-H2-DM (clone 2E5a; Fisher Scientific; cat. no. 552405), anti-rat IgG1 (FITC; clone RG11/39.4; Fisher Scientific; cat. no. BDB553892), and anti-Nur77 (PE; clone 12.14; Fisher Scientific; cat. no. 5011028).

Techniques: Flow Cytometry, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nature immunology

Article Title: Strength of tonic T cell receptor signaling instructs T follicular helper fate decisions

doi: 10.1038/s41590-020-0781-7

Figure Lengend Snippet: a , Outline of the Scn5a construct that was used to generate the F/S/F- Scn5a mouse, previously described , and the breeding scheme of the LLO118 + Scn5a + Cd4-creErt2 + line. Upon tamoxifen treatment, ectopic Scn5a expression in peripheral CD4 + T cells can be detected by GFP expression. b, Representative flow plots depicting GFP detection in CD4 + T cells from LLO118 + Scn5a + Cd4-creErt2 + mice 7 days post-tamoxifen treatment. Both the LLO118 + Scn5a + Cd4-creErt2 + mouse and its littermate control were treated with tamoxifen. c-e , Flow cytometry analysis of CD5, Ly6C, and TCRβ MFI in LLO118 GFP + and GFP − T cells from LLO118 + Scn5a + Cd4-creErt2 + mice 7 days post-tamoxifen treatment. Data points are the paired GFP + and GFP − populations from individually treated mice; three independent experiments (n=7). f , Analysis of LLO118 + Scn5a + Cd4-creErt2 + GFP + T cell expansion during a primary immune response. LLO118 + Scn5a + Cd4-creErt2 + and LLO118 + Scn5a − Cd4-creErt2 + mice were treated with tamoxifen and 7 days later CD4 + T cells were enriched and transferred into Tcra −/− recipients. The following day, recipients were immunized with NP-LLO LT -N. Graphs show the frequency of GFP + cells in the LLO118 populations immediately prior to the transfer (unstimulated, left graph) and 7 days post-immunization (d7, right graph). Three independent experiments (n=7 for LLO118 + Scn5a + Cd4-creErt2 + and n=9 for LLO118 + Scn5a − Cd4-creErt2 + ). Mean ± SEM are shown. g , In the same experiments as (f), in vivo -activated LLO118 + Scn5a + Cd4-creErt2 + cells were also analyzed at day 7 post-immunization for the frequency of Tfh cells within the GFP + and GFP − populations. ****p < 0.0001, **p < 0.01, *p < 0.05 . Paired t test or Wilcoxon matched-pairs signed rank test for nonnormally distributed data (c-e, g). Unpaired t test or Mann-Whitney test for nonnormally distributed data (f).

Article Snippet: The following antibodies were used for intracellular flow cytometry analysis: anti-Bcl6 (PE; clone BCL-DWN; Fisher Scientific; cat. no. 501122326; 1:100 dilution), anti-Tbet (PE; clone eBio4B10; Fisher Scientific; cat. no. 5010893), anti-GATA-3 (PE-Cy7; clone TWAJ; Fisher Scientific; cat. no. 501129305), anti-RORγt (BV421; clone Q31-378; BD Biosciences; cat. no. 562894), anti-IFN-γ (FITC; clone XMG1.2; Biolegend; cat. no. 505806), anti-IL-4 (Biotin; clone BVD6-24G2; BD Biosciences; cat. no. 554390; 1:100 dilution), anti-IL-21 (PE; clone FFA21; Fisher Scientific; cat. no. 5011172; 1:100 dilution), anti-H2-DM (clone 2E5a; Fisher Scientific; cat. no. 552405), anti-rat IgG1 (FITC; clone RG11/39.4; Fisher Scientific; cat. no. BDB553892), and anti-Nur77 (PE; clone 12.14; Fisher Scientific; cat. no. 5011028).

Techniques: Construct, Expressing, Control, Flow Cytometry, In Vivo, MANN-WHITNEY