BRD4 positively regulates FSP1 expression in GCB-DLBCL. (A-B) Various GCB-DLBCL cell lines were treated with 0.25 μM (A) or the indicated concentrations (B) of JQ1 for 24 hours. AIFM2 transcript levels were assessed by qPCR. SDHA served as reference gene. (C) FSP1 and GPX4 protein expression was analyzed by immunoblotting in various GCB-DLBCL cell lines treated with solvent, JQ1 or I-BET151 for 24 hours. GAPDH served as loading control. (D) In SU-DHL-6 and OCI-Ly7 cells, BRD4 expression was silenced using 3 independent shRNAs. FSP1 and GPX4 protein levels were visualized by immunoblotting. GAPDH served as loading control. (E) Chromatin immunoprecipitation analysis of BRD4 binding to AIFM2 and SLC7A11 promoter regions. For each gene, 2 independent promoter-specific (#1 and #2) and 1 upstream (upstr.) primer as internal negative control are shown. (F) The indicated GCB-DLBCL cell lines were treated with the CDK9 inhibitor LDC067 for 24 hours and analyzed for FSP1 expression by immunoblotting. GAPDH served as loading control. Error bars correspond to the mean ± SD. Data are representative of at least 3 (A-C) or 2 (D-F) independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.