Figure 1 from BET Inhibition Synergizes with RLR Signaling to Enhance Tumor Immunogenicity and T-cell Recognition

Abstract

BETi pretreatment sensitizes tumor cells to pIC-induced inflammation. A, Experimental setup for BETi pretreatment and pIC transfection of tumor cells. B, Protein abundance of phosphorylated IRF3 and (C) TBK1 in A375 cells after 72 hours of JQ1 or DMSO pretreatment and pIC transfection for 24 hours assessed by Western blot. n = 5 biological replicates. A representative Western blot is depicted in (D). E, Expression heatmap of selected genes from qPCR after 72 hours of JQ1 treatment and subsequent pIC transfection for 24 hours in ANRU, (F) KADA, and (G) A375. Data are represented as mean fold change from DMSO. n = 3 biological replicates/cell line. H, Bar plot displaying gene expression of selected genes from G (log2 fold change from DMSO). I, CXCL10 and (J) IFNβ concentration in supernatants from treated A375, ANRU, and BEHA cells. n = 3 biological replicates/cell lines. K, Cell surface expression of HLA-I was assessed by flow cytometry after 48 hours of pIC stimulation preceded by either DMSO or JQ1 treatment in four melanoma cell lines. n ≥ 3 biological replicates/cell line. L, Representative histogram plot for A375 from (G). M, Cell surface expression of HLA-I assessed by flow cytometry after 48 hours of pIC stimulation preceded by either DMSO or JQ1 treatment in the colorectal cancer cell line COLO320. n = 3 biological replicates. N, Cell surface expression of HLA-I in A375 after JQ1/pIC in the presence or absence of an IFNα/βR1-blocking antibody (2 µg/mL). n = 3 biological replicates. O, HLA-I expression in ANRU cells upon concomitant treatment with JQ1 and/or nontargeting control siRNA (NTC) or siRNA targeting TLR3, RIG-I, or MDA-5, prior to transfection with pIC. n = 3 biological replicates. Error bars represent mean ± standard deviation. Statistical analysis was performed using paired one- or two-way ANOVA with Holm–Šidák correction for multiple comparisons. P values <0.05 are indicated above the relevant comparisons. geoMFI, geometric mean fluorescence intensity. [A, Created in BioRender. Wickstrom, S. (2026) https://BioRender.com/npulau2.]