5-LOX sensitizes GCB DLBCL cells to DMF-induced ferroptosis. (A) Protein expression of 5-LOX, FSP1, and system xc− in the indicated cell lines was visualized by immunoblot analysis. ERK1/2, Tom20, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading controls. (B) ALOX5 transcript levels in DLBCL or myeloid cell lines were quantified by quantitative polymerase chain reaction and normalized to the expression in U937 cells. SDHA served as reference gene. (C) Immunohistochemical staining of 5-LOX in human GCB and ABC DLBCL biopsies. Scale bars, 50 μm. (D) Quantification of nuclear 5-LOX staining in DLBCL biopsies (GCB DLBCL, n = 89; ABC DLBCL, n = 64). (E) The indicated ABC DLBCL cell lines were treated with 5 μM sotrastaurin (STN), 0.1 μM ibrutinib, 4 μM GS-9973, or 1 μM dasatinib for 24 hours. Transcript levels of ALOX5 and TNFAIP3 were quantified and normalized to the solvent control. SDHA served as reference gene. (F) Quantification of ALOX5 and TNFAIP3 messenger RNA (mRNA) expression in the indicated GCB DLBCL cell lines activated for 8 hours with PMA/ionomycin (P/I). SDHA served as reference gene. (G) The survival of SU-DHL-6 cells treated with 20 μM DMF alone or in combination with 10 μM zileuton, 2.5 μM CJ-13610, 5 μM CAY10649, or 10 μM MK-886 was assessed after 24 hours by MTS assay. (H) Quantification of lipid peroxidation in DMF-treated control and ALOX5-silenced DOHH2 cells. The mean fluorescence intensity (MFI) of oxidized BODIPY C11 in DMF-treated cells was normalized to the MFI of the respective solvent-treated samples. Error bars correspond to the mean ± standard deviation. Data are representative of ≥2 (E) or ≥3 (A-B,F-H) independent experiments. Statistical significance was calculated using the Student t test. **P < .01, ***P < .001. sh, short hairpin.