pan bet inhibitor jq1  (Tocris)

Journal: bioRxiv

Article Title: Unraveling AMPK and BET regulation of immune checkpoint biology: implications for personalized medicine

doi: 10.64898/2026.01.26.701869

Figure Lengend Snippet: BET proteins differentially regulate TIM-3, TIGIT, PD-1 and CTLA-4 expression on both T cells and NK cells. Purified PBMCs were activated by plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (2 µg/ml) ex vivo at 37□ for 72h and treated with pan-BET inhibitor JQ1 (400 nM) or BRD4-selective PROTAC degrader MZ-1 (50 nM), respectively, for the last 48h of 72h. Cell surface fluorescence staining and flow cytometry analysis were performed to measure expression changes of TIM-3, TIGIT, PD-1, CTLA-4 on CD4+, CD8+ T cells and NK cells. (A-C, G-I) Representative histograms show expression shifts, comparing stimulated-only cells with stimulated plus JQ1 or plus MZ-1 inhibitors. X-axis: intensity of fluorescence signal. Y-axis: number of events (D-F, J-L) Corresponding bar graphs show mean fluorescence intensity (MFI) of markers on T and NK cells, comparing stimulated cells to stimulated plus JQ1 or plus MZ-1. Two-tailed t-test and Mann Whitney U test determined statistical significance. (N=6; ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001)

Article Snippet: Pan-BET inhibitor JQ1 (Tocris), BRD4-selective PROTAC degrader MZ-1 (Tocris), and AMPK inhibitor Compound C (Tocris) were dissolved in DMSO to final concentrations of 400 nM, 50 nM and 5 μM respectively.

Techniques: Expressing, Purification, Ex Vivo, Fluorescence, Staining, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Unraveling AMPK and BET regulation of immune checkpoint biology: implications for personalized medicine

doi: 10.64898/2026.01.26.701869

Figure Lengend Snippet: PBMCs were activated ex vivo by anti-CD3 and anti-CD28 at 37□ for 72h as above. Cell surface fluorescence staining and flow cytometry were performed to measure expression of TIM-3, TIGIT, PD-1 and CTLA-4 on both T and NK cells. Experimental groups were: stimulated plus Compound C (2.5μM), stimulated plus JQ1/MZ1 (JQ1: 400nM, MZ1: 50nM), stimulated plus Compound C and JQ1/MZ1. (A-C, G-I) Histograms combined the expression shifts of four markers in cells treated by different condition. X-axis: intensity of fluorescence signal. Y-axis: number of events. (D-F, J-L) Bar graphs show mean fluorescence intensity (MFI) of TIM-3, TIGIT, PD-1 and CTLA-4, for stimulated cells treated with Compound C, JQ1/MZ1 respectively and combined. Two-tailed t-test and Mann Whitney U test was used to measure statistical significance. (ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001)

Article Snippet: Pan-BET inhibitor JQ1 (Tocris), BRD4-selective PROTAC degrader MZ-1 (Tocris), and AMPK inhibitor Compound C (Tocris) were dissolved in DMSO to final concentrations of 400 nM, 50 nM and 5 μM respectively.

Techniques: Ex Vivo, Fluorescence, Staining, Flow Cytometry, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Oncogene

Article Title: Targeting non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex improves the efficacy of HEDGEHOG pathway inhibition in melanoma

doi: 10.1038/s41388-021-01783-9

Figure Lengend Snippet: A qPCR of BRD4 and GLI1 after BRD4 silencing with two independent shRNAs. B Western blot analysis of BRD4 and GLI1 in melanoma cells transduced as indicated. HSP90 was used as loading control. C ChIP-qPCR of BRD4 occupancy on GLI1 promoter in SSM2c treated with vehicle (DMSO) or 100 nM JQ1 for 18 h. Data are presented as % of input and are expressed as fold over IgG control ± SEM ( n = 3). D Quantification of dual-luciferase assay in SSM2c melanoma cells treated with DMSO or increasing concentrations of JQ1. Relative luciferase activities were firefly/Renilla ratios, with the level induced by the vehicle equated to 1 ( n = 3). E qPCR of GLI1 in three melanoma cell lines treated with DMSO or increasing concentrations of JQ1. F WB of BRD4 and GLI1 in melanoma cells treated with JQ1 as indicated. HSP90 was used as loading control. G Histogram of melanoma cell viability in cells transduced with LV-c or LV-shBRD4 and treated with DMSO or increasing concentrations of JQ1 or MZ1 ( n = 3). H Quantification of dual-luciferase assay in SSM2c cells treated with DMSO or increasing concentrations of MZ1 ( n = 3). I WB of BRD4 and GLI1 in melanoma cells treated with DMSO or increasing concentrations of MZ1. HSP90 was used as loading control. Data are presented as mean ± SEM. P values were calculated by two-tailed unpaired Student’s t test ( A , D , E , G , H ) or one-way ANOVA with Tukey’s test ( C ). *, p < 0.05; **, p < 0.01; ***, p < 0.0001.

Article Snippet: The pan-BET inhibitor JQ1 (Catalog No. S7110, purity ≥99%) was purchased from Selleckchem (Munich, Germany).

Techniques: Western Blot, Control, ChIP-qPCR, Luciferase, Transduction, Two Tailed Test

Journal: Oncogene

Article Title: Targeting non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex improves the efficacy of HEDGEHOG pathway inhibition in melanoma

doi: 10.1038/s41388-021-01783-9

Figure Lengend Snippet: A Co-IP of SOX2 and BRD4 in SSM2c lysates untreated or exposed to 25 U/ml DNase or 200 μg/ml ethidium bromide (EtBr). Input was 5%. B ChIP-qPCR of SOX2 occupancy at GLI1 promoter in SSM2c LV-c treated with vehicle (DMSO), JQ1 (100 nM) or MZ1 (125 nM), or LV-shBRD4. C Western blot of BRD4, SOX2 and GLI1 in SSM2c cells transduced with LV-c or LV-shBRD4 (upper panel) or treated with DMSO, JQ1 or MZ1 for 24 h (lower panel). D – E Dual-luciferase assay in SSM2c cells transduced with LV-c or LV-shBRD4 ( D ) or treated with increasing concentrations of MZ1 ( E ). It shows that Mut2 prevents BRD4 from transactivating the −584/−133 fragment of GLI1 promoter in absence of a functional SOX2-BS (Mut2) ( n = 3). F – G Dual-luciferase assay in SSM2c cells transduced with LV-c or LV-shBRD4 in presence or absence of SOX2 ( F ) or treated with increasing concentrations of MZ1 in presence or absence of SOX2 ( G ) as indicated ( n = 3). H Venn diagram showing that the distribution of cells expressing GLI1, SOX2 , and/or BRD4 is different between normal human neonatal epidermal melanocytes (NHEM, left) and patient-derived melanoma xenografts (M15, right). Single cell RNA-seq data were filtered to the 80th percentile of high gene expression, and number of cells expressing one, two, or all three of these genes was quantified. The green circle represents number of cells expressing BRD4 , the blue circle represents SOX2 , and the red circle represents GLI1 . The cutoff values for expression representing the 80th percentile in SOX2, GLI1 , and BRD4 were 0.06783216, 0.6914666, and 0.6897033, respectively. In ( B , D , E , F , G ), data are presented as mean ± SEM. P values were calculated by one-way ANOVA with Tukey’s test ( B ) or two-tailed unpaired Student’s t test ( D – G ). *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns not significant.

Article Snippet: The pan-BET inhibitor JQ1 (Catalog No. S7110, purity ≥99%) was purchased from Selleckchem (Munich, Germany).

Techniques: Co-Immunoprecipitation Assay, ChIP-qPCR, Western Blot, Transduction, Luciferase, Functional Assay, Expressing, Derivative Assay, RNA Sequencing, Gene Expression, Two Tailed Test