Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: a The protein levels of Smyd3 and Shcbp1 in normal mouse mammary glands, and tumors initiated with HP5712 cells in 3 months old FVB virgin mice as shown by Western blots. b The protein levels of Smyd3 and Shcbp1 in HP5712 cells expressing sgSmyd3, OE-Smyd3, or OE-Smyd3/sgShcbp1 by Western blots. Representative tumor images ( c ) and tumor weight plots ( d ) at day 32 from FVB virgin mice implanted with HP5712 parental (HP tumors), OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells at 1X10 6 cells per mammary fat pad (n = 8 mice/group). e The plot of relative spleen weight from the same cohort of mice in c and d . f The protein levels of Smyd3, H3K4me3, and Shcbp1 in tumors initiated with HP5712 parental, OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells by Western blots. g The protein levels of pMek, pErk, and Kras in tumors initiated with HP5712 parental, OE-Smyd3-HP5712, sgSmyd3-HP5712, and OE-Smyd3/sgShcbp1-HP5712 cells as shown by Western blots. h tSNE visualized immune cells from normal FVB mouse mammary glands (FVB MG, n = 6 mice), tumors implanted with parental HP5712 cells (HP Vector Ctr), HP-OE-Smyd3, HP-OE-Smyd3/sgShcbp1, and HP-sgSmyd3 from FVB mice (n = 6 mice/group) by CyTOF analysis. Quantifications of PMN-MDSCs and M-MDSCs ( i ), CD4+ and CD8 + T cells ( j ) in CD45+ immune cell populations from the same cohorts of mice in h (n = 3 biological independent samples/per group). k , l The WT-T cells were activated with CD3 and CD28 antibody. Co-cultured the activated T cells without or with MDSCs from the mice implanted with HP parental cells, HP cells expressing Smyd3, or sgSmyd3, or OE-Smyd3/sgShcbp1 for 72 hours and examined T cells proliferation status ( k ) and quantifications ( l ) of data in k (n = 3 mice/group). Representative images of CD8+/PD1+ double positive cells in mammary tissues from the mice with the genotypes of HP-Ctr, HP-OE-Smyd3, HP-sgSmyd3, and HP-OE-Smyd3/sgShcbp1 ( m ) and quantifications of CD8+/PD1 double positive cells from mammary tissues ( n ) in these mice (n = 4 mice/group, and at least 20 images for each sample were counted). Scale bar: 20 μm. arrow heads point to PD1/CD8 positive cell. o Expressions of PD1 from the spleen with implantation of parental HP cells, HP cells with OE-Smyd3, HP-sgSmyd3, and HP-OE-Smyd3/sgShcbp1 in FVB mice as determined by qPCR. p Summary of Smyd3-Shcbp1oncogenic signals shape the TIME.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Western Blot, Expressing, Plasmid Preparation, Cell Culture

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Representative tumor images ( a ), and tumor weight plots ( b ) at day 22 from FVB mice implanted with HP5712 cells at 1×10 6 cells per mammary fat pad, and treatment with PBS, PD1, trametinib (Tra), and PD1+Tra (n = 8 mice/group). c The plot of tumor volume in the processes of treatment in ( a , b ). d The plot of relative spleen weight from the same cohort of mice in a , b . Quantifications of PMN-MDSCs and M-MDSCs ( e ), CD4+ and CD8 + T cells ( f ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D12 (n = 3 mice/group). Quantifications of PMN-MDSCs and M-MDSCs ( g ), CD4+ and CD8 + T cells ( h ) in CD45+ immune cell populations from the same cohorts of mice in a , b by CyTOF analysis at D22 (n = 3 mice/group). i , j Tumor images from the HP Ctr mice, HP mice treated with αPD1 and Tra, and HP mice treated with αPD1 and Tra with depletion of T cell using CD8 antibody ( i ). The plot of tumor weight ( j ) in the same cohort of mice in ( i ) (n = 8 mice in each group). The protein levels of Smyd3 and Shcbp1 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( k ) and D22 ( l ) as shown by Western blots. The protein levels of pMek, pErk, Kras, and Grb2 in tumors initiated with HP5712 cells and treatment with PBS, αPD1, Tra, and αPD1+Tra in FVB mice at D12 ( m ) and D22 ( n ) as shown by Western blots. Expressions of Smyd3 ( o ) and Shcbp1 ( p ) in G600 cells with the treatment of E2, Tra, and E2 together with Tra at 1 hour, 2 hours, 4 hours, and 24 hours as determined by qPCR.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Oncogenic activation of SMYD3-SHCBP1 promotes breast cancer development and is coupled with resistance to immune therapy

doi: 10.1038/s41419-025-07570-8

Figure Lengend Snippet: Antibodies used for ChIP, IHC, IF, WB and in vivo mouse study.

Article Snippet: In Vivo anti-mouse CD8a , BioX Cell , BP0117 , , .

Techniques: Immunohistochemistry-IF, In Vivo

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: eLife

Article Title: CD8+ T cell self-tolerance permits responsiveness but limits tissue damage

doi: 10.7554/elife.65615

Figure Lengend Snippet: Figure 2. Differences in the magnitude of the response to Trp2 immunization in wild-type (WT) and Dct-/- mice. Mice were primed with TriVax (50 mg each of Trp2 and B8R peptides; A–E). The number (A) or percent (B) of splenic Trp2/Kb or B8R/Kb-specific cells was assessed at day 7. (C, D) The tetramer fluorescence intensity of splenic Trp2/Kb-specific cells was compared. (E) Gating for dual Trp2/Kb tetramer-positive CD8+ (samples were not enriched for Trp2/Kb-specific cells). (F) The frequency of the indicated splenic population expressing PD-1 is shown. Data in A and B are compiled from more than three experiments. Data in C–F are representative of three or more similar experiments. Squares indicate male animals. *p<0.05, ****p<0.0001 by unpaired t test (C) or one- way ANOVA with Sidak’s multiple comparisons test (A, B, F). The online version of this article includes the following source data and figure supplement(s) for figure 2:

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Poly(I:C) Invivogen Cat. #: vac-pic Antibody InVivoMAb anti-mouse CD40 BioXCell Cat. #: BE0016-2; RRID:AB_1107647 Clone FGK4.5 Peptide, recombinant protein Trp2 peptide New England Peptide Trp2180-188 H2NSVYDFFVWL-OH Chemical compound, drug 1-Fluoro-2,4dinitrobenzene (DNFB) Sigma Aldrich Cat. #: D-1529 Antibody Anti-mouse CD8a FITC Tonbo Biosciences Cat. #: 35-0081; RRID:AB_2621671 Clone 53-6.7 Antibody Anti-mouse CD8a vf450 Tonbo Biosciences Cat. #: 75-0081; RRID:AB_2621931 Clone 53-6.7 Antibody Anti-mouse CD8a PerCP-Cy5.5 Tonbo Biosciences Cat. #: 65-0081; RRID:AB_2621882 Clone 53-6.7 Antibody Anti-mouse CD8a APCef780 eBioscience Cat. #: 47-0081-80; RRID:AB_1272221 Clone 53-6.7 Antibody Anti-mouse CD4 BV605 BioLegend Cat. #: 100548; RRID:AB_2563054 Clone RM4-5 Antibody Anti-mouse CD4 PE-Cy7 Tonbo Biosciences Cat. #: 60-0042; RRID:AB_2621829 Clone RM4-5 Antibody Anti-mouse CD44 BV786 BD Biosciences Cat. #: 563736; RRID:AB_2738395 Clone IM7 Antibody Anti-mouse CD44 FITC eBioscience Cat. #: 11-0441-85; RRID:AB_465046 Clone IM7 Antibody Anti-mouse CD44 rf710 Tonbo Biosciences Cat. #:80-0441; RRID:AB_2621985 Clone IM7 Antibody Anti-mouse CD45.2 FITC Tonbo Biosciences Cat. #: 35-0454; RRID:AB_2621692 Clone 104 Antibody Anti-mouse CD45.1 PE-Cy7 Tonbo Biosciences Cat. #: 60-0453; RRID:AB_2621850 Clone A20 Antibody Anti-mouse CD90.1 ef450 eBioscience Cat. #: 48-0900-82; RRID:AB_1272254 Clone HIS51 Antibody Anti-mouse CD90.2 PE-Cy7 Tonbo Biosciences Cat. #: 60-0903; RRID:AB_2621857 Clone 30-H12 Antibody Anti-mouse MHC Class II (I-A/I-E) APC ef780 Thermo Fisher Scientific Cat. #: 47-5321-82; RRID:AB_1548783 Clones M5/114.15.2 Antibody Anti-mouse MHC Class II (I-A/I-E) BV510 BioLegend Cat. #: 107635; RRID:AB_2561397 Clones M5/114.15.2 Antibody Anti-mouse F4/80 BV510 BioLegend Cat. #: 123135; RRID:AB_2562622 Clone BM8 Antibody Anti-mouse F4/80 APC ef780 Thermo Fisher Scientific Cat. #: 47-4801-82; RRID:AB_2735036 Clone BM8 Antibody Anti-mouse CD122 BV421 BD Biosciences Cat. #: 562960; RRID:AB_2737918 Clone TM-b1 Antibody Anti-human Granzyme B PE Invitrogen/Thermo Cat. #: GRB04; RRID:AB_2536538 Clone GB11 Antibody Anti-mouse IFN-g PE-Cy7 Tonbo Biosciences Cat. #: 60-7311-U100; RRID:AB_2621871 Clone XMG1.2 Antibody Anti-mouse TNFa APC eBioscience Cat. #: 17-7321-81; RRID:AB_469507 Clone MP6-XT22

Techniques: Fluorescence, Expressing

Journal: eLife

Article Title: CD8+ T cell self-tolerance permits responsiveness but limits tissue damage

doi: 10.7554/elife.65615

Figure Lengend Snippet: Figure 5. Wild-type (WT) Trp2/Kb-specific cells show proliferative defects in the early effector phase. Trp2/Kb-specific CD8+ T cells were isolated from WT and Dct-/- mice on day 3 after TriVax and submitted for scRNA-seq. After initial processing, the WT and Dct-/- datasets were merged and further analyzed. (A) Uniform manifold approximation and projection (UMAP) representation of gene expression from merged datasets determined using Seurat; each dot represents one cell. Clusters are indicated by color. (B) Cells from the Dct-/- sample are shown on the left and cells from the WT Figure 5 continued on next page

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Poly(I:C) Invivogen Cat. #: vac-pic Antibody InVivoMAb anti-mouse CD40 BioXCell Cat. #: BE0016-2; RRID:AB_1107647 Clone FGK4.5 Peptide, recombinant protein Trp2 peptide New England Peptide Trp2180-188 H2NSVYDFFVWL-OH Chemical compound, drug 1-Fluoro-2,4dinitrobenzene (DNFB) Sigma Aldrich Cat. #: D-1529 Antibody Anti-mouse CD8a FITC Tonbo Biosciences Cat. #: 35-0081; RRID:AB_2621671 Clone 53-6.7 Antibody Anti-mouse CD8a vf450 Tonbo Biosciences Cat. #: 75-0081; RRID:AB_2621931 Clone 53-6.7 Antibody Anti-mouse CD8a PerCP-Cy5.5 Tonbo Biosciences Cat. #: 65-0081; RRID:AB_2621882 Clone 53-6.7 Antibody Anti-mouse CD8a APCef780 eBioscience Cat. #: 47-0081-80; RRID:AB_1272221 Clone 53-6.7 Antibody Anti-mouse CD4 BV605 BioLegend Cat. #: 100548; RRID:AB_2563054 Clone RM4-5 Antibody Anti-mouse CD4 PE-Cy7 Tonbo Biosciences Cat. #: 60-0042; RRID:AB_2621829 Clone RM4-5 Antibody Anti-mouse CD44 BV786 BD Biosciences Cat. #: 563736; RRID:AB_2738395 Clone IM7 Antibody Anti-mouse CD44 FITC eBioscience Cat. #: 11-0441-85; RRID:AB_465046 Clone IM7 Antibody Anti-mouse CD44 rf710 Tonbo Biosciences Cat. #:80-0441; RRID:AB_2621985 Clone IM7 Antibody Anti-mouse CD45.2 FITC Tonbo Biosciences Cat. #: 35-0454; RRID:AB_2621692 Clone 104 Antibody Anti-mouse CD45.1 PE-Cy7 Tonbo Biosciences Cat. #: 60-0453; RRID:AB_2621850 Clone A20 Antibody Anti-mouse CD90.1 ef450 eBioscience Cat. #: 48-0900-82; RRID:AB_1272254 Clone HIS51 Antibody Anti-mouse CD90.2 PE-Cy7 Tonbo Biosciences Cat. #: 60-0903; RRID:AB_2621857 Clone 30-H12 Antibody Anti-mouse MHC Class II (I-A/I-E) APC ef780 Thermo Fisher Scientific Cat. #: 47-5321-82; RRID:AB_1548783 Clones M5/114.15.2 Antibody Anti-mouse MHC Class II (I-A/I-E) BV510 BioLegend Cat. #: 107635; RRID:AB_2561397 Clones M5/114.15.2 Antibody Anti-mouse F4/80 BV510 BioLegend Cat. #: 123135; RRID:AB_2562622 Clone BM8 Antibody Anti-mouse F4/80 APC ef780 Thermo Fisher Scientific Cat. #: 47-4801-82; RRID:AB_2735036 Clone BM8 Antibody Anti-mouse CD122 BV421 BD Biosciences Cat. #: 562960; RRID:AB_2737918 Clone TM-b1 Antibody Anti-human Granzyme B PE Invitrogen/Thermo Cat. #: GRB04; RRID:AB_2536538 Clone GB11 Antibody Anti-mouse IFN-g PE-Cy7 Tonbo Biosciences Cat. #: 60-7311-U100; RRID:AB_2621871 Clone XMG1.2 Antibody Anti-mouse TNFa APC eBioscience Cat. #: 17-7321-81; RRID:AB_469507 Clone MP6-XT22

Techniques: Isolation, Gene Expression

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A IOD of ACAT2 expression in CC tissues and adjacent tissues was examined using immunohistochemical staining ( n = 47 biologically independent samples). IOD of DHCR7 B and MSMO1 C expression in CC patients with high ( n = 27 biologically independent samples) or low ( n = 20 biologically independent samples) expression of ACAT2 was examined using immunohistochemical staining. The number of activated CD8 T cells (CD8A + GZMB + ) D or activated NK cells (CD56 + GZMB + ) E infiltrated in the tumor tissues of patients with high ( n = 27 biologically independent samples) and low ACAT2 ( n = 20 biologically independent samples) expression was detected. Data represent mean ± SEM. Statistical analysis was performed using the paired A or unpaired ( B – E ) t-test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: ACAT2 expression in HCeEpiC and CC cell lines was examined using RT-qPCR A and Western blot analysis B ( n = 5 independent experiments). C ACAT2, DHCR7, and MSMO1 expression in CC cells after infection with Scramble-sh, ACAT2-sh #1, and ACAT2-sh #2 was examined using Western blot analysis ( n = 5 independent experiments). D Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in CC cells ( n = 5 independent experiments). The proliferation of CC cells was examined using CCK8 ( E ) and colony formation assays F (n = 5 independent experiments). G CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 T cells for 6 h, respectively, and the death of CC cells was detected ( n = 5 independent experiments). H IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , B ) or two-way ( C - H ) ANOVA, followed by Tukey’s multiple comparisons test ( A – H ).

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A ACAT2 knockdown efficiency in U14 cells was examined using western blot analysis ( n = 10 independent experiments). B Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells (n = 10 animals). C The images and weight of the tumors harvested on day 21 ( n = 10 animals). D Protein expression of ACAT2, MSMO1, DHCR7, and PCNA in transplanted tumors was examined using western blot analysis ( n = 10 animals). E Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in transplanted tumors ( n = 10 animals). The gating strategy for GZMB + NK cells and CD8 + T cells F and quantification G were analyzed using flow cytometry ( n = 10 animals). H Survival of mice over 60 days after subcutaneous inoculation of U14 cells was analyzed using the log-rank test ( n = 20 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , C , E , G ) or two-way ( B , D ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: The proliferation of CC cells was examined using CCK8 A and colony formation assays B ( n = 5 independent experiments). C CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 + T cells, and the death of CC cells was detected ( n = 5 independent experiments). D IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). E TGF-β1 released by CC cells was examined using ELISA ( n = 5 independent experiments). F PD-L1 expression levels in CC cells were observed using immunofluorescence staining ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the two-way ( A – F ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells ( n = 5 animals). B The images and weight of the tumors harvested on day 21 ( n = 5 animals). The gating strategy for GZMB + NK cells and CD8 + T cells C and quantification D were analyzed using flow cytometry ( n = 5 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( B , D ) or two-way A ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: BRD7 Inhibited Immune Escape in Nasopharyngeal Carcinoma via Inhibiting PD-L1 Expression

doi: 10.7150/ijbs.103703

Figure Lengend Snippet: BRD7 downregulated PD-L1 expression and enhanced the cytotoxicity of T lymphocytes against NPC cells. (A) CIBERSORT determined the proportion of immune cell populations in NPC. (B) CD8 + T cell infiltration plays a vital role in NPC. (C) Correlation analysis between the expression of BRD7 and immune cells abundance. (D) Correlation analysis between BRD7 expression level and immune cell subtypes in GSE102349 of NPC. (E) Clonogenic assays of 5-8F and CNE2 cells stably transfected with BRD7 overexpression and knockdown or empty vector plasmids with or without T cells co-culture and PD-L1 antibody incubation. Atezolizumab: the PD-L1 antibody. (F) CCK-8 assay of 5-8F and CNE2 cells stably transfected with BRD7 overexpression and knockdown or empty vector plasmids with or without T cells co-culture and PD-L1 antibody incubation. Absorbance values were detected at 450 nm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (G) Flow cytometry detecting the apoptosis ratio of CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 overexpression or empty vector plasmids with or without PD-L1 antibody incubation. (H) Flow cytometry detecting the apoptosis ratio of CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 knockdown or empty vector plasmids. (I) Flow cytometry detecting the ratio of PD-1 + CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 overexpression or empty vector plasmids with or without PD-L1 antibody incubation. (J) Flow cytometry detecting the ratio of PD-1 + CD8 + T cells in T cells co-cultured with 5-8F or CNE2 cells stably transfected with BRD7 knockdown or empty vector plasmids. (K) ELISA detecting the IFN-γ content in the culture medium of T cells co-cultured with 5-8F cells stably transfected with BRD7 overexpression or empty vector plasmids and CNE2 cells stably transfected with BRD7 knockdown or empty vector plasmids with or without PD-L1 antibody incubation for 24 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: T lymphocytes and tumor cells were cocultured at a ratio of 10:1 for 24 h. T cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit (Biosharp, China), APC Anti-Human CD3 Antibody (Elabscience, China), PerCP Anti-Human CD8a Antibody (Elabscience, China), and FITC Anti-Human CD279/PD-1 Antibody (Elabscience, China) according to the instructions.

Techniques: Expressing, Stable Transfection, Transfection, Over Expression, Knockdown, Plasmid Preparation, Co-Culture Assay, Incubation, CCK-8 Assay, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: BRD7 Inhibited Immune Escape in Nasopharyngeal Carcinoma via Inhibiting PD-L1 Expression

doi: 10.7150/ijbs.103703

Figure Lengend Snippet: BRD7 could inhibit the immune escape of NPC. (A) Design of the experiment in vivo . (B) Images of the appearance of subcutaneous tumors in mice. n = 5 per group. (C) Statistical analysis of subcutaneous tumor weight. n = 5 per group. (D) Tumor growth curve. n = 5 per group. (E) Relative BRD7 and PD-L1 mRNA levels measured by q-PCR in tumor tissues. n = 5 per group. (F) Western blot analysis of BRD7, PD-L1, and PI3K/AKT pathway molecules in tumor tissues. (G) Representative images of immunohistochemical staining for BRD7, PD-L1, PI3K/AKT pathway molecules, and CD8 expression in tumor tissues. The scale bar is 20 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: T lymphocytes and tumor cells were cocultured at a ratio of 10:1 for 24 h. T cells were stained with an Annexin V-FITC/PI Apoptosis Detection Kit (Biosharp, China), APC Anti-Human CD3 Antibody (Elabscience, China), PerCP Anti-Human CD8a Antibody (Elabscience, China), and FITC Anti-Human CD279/PD-1 Antibody (Elabscience, China) according to the instructions.

Techniques: In Vivo, Western Blot, Immunohistochemical staining, Staining, Expressing