BET inhibitor-mediated reduction of FSP1 expression promotes ferroptosis. (A) The indicated GCB-DLBCL cell lines were treated with solvent, iFSP1, DMF, or with a combination of the compounds for 2 hours. Lipid peroxidation was quantified by flow cytometry. (B) DMF-induced lipid peroxidation was analyzed in control or AIFM2-silenced DOHH2 cells. (C) SU-DHL-6 cells were treated with DMF alone (left panel) or in combination with iFSP1 (right panel), as indicated. After 24 hours, cell numbers were determined, and the combination treatments were normalized to the DMF single treatment. The combination index (CI) for treatment with 7.5 μM DMF and 2.5 μM iFSP1 was 0.38. (D) Lipid peroxidation was quantified by flow cytometry in control or FSP1-FLAG overexpressing DOHH2 cells after combination treatment with JQ1 and DMF. The MFI of oxidized BODIPY C11 in treated cells was normalized to the MFI of the solvent control. (E) DOHH2 cells were transduced with a control or an FSP1-FLAG encoding plasmid. Cell numbers upon incubation with DMF (left panels) or DMF and JQ1 (right panels) were determined and the combinatorial treatments were normalized to the DMF single treatment. (F) DOHH2 and WSU-DLCL-2 cells were treated with iFSP1 alone or in combination with the indicated JQ1 concentrations. Cell survival was assessed after 48 hours. Error bars correspond to the mean ± SD. Data are representative of at least 3 (A,C-E) or 2 (B,F) independent experiments. ∗∗P < .01; ∗∗∗P < .005. MFI, mean fluorescence intensity.