IRE1 expression levels and activity are regulated by histone methyltransferase EZH2. (A) Karpas422 cells treated for 5 days with 2.5 μM of the EZH2 inhibitor GSK343 or vehicle were analyzed by ChIP for H3K27me3 promoter marks on MyoD and IRF4 (EZH2-dependent), GAPDH (negative control), and IRE1 (mean and SD of technical triplicates of 1 representative experiment of 3). (B) Representative ABC and GCB DLBCL cell lines treated with GSK343 or vehicle were probed by immunoblot for IRE1, PERK, H3K27me3, H3 total, and tubulin (Tbl). IRE1 protein levels were quantified and plotted (right panel). Paired Student t test (nontreated vs treated); *P ≤ .05. (C) Representative GCB DLBCL cell lines were treated for 5 days with 2.5 μM EZH2 inhibitor GSK343. Then, 5 μg/mL tunicamycin (TM) was added to the cells for 6 hours. Immunoblots were revealed using antibodies against XBP1s, IRE1, PERK, ATF4, H3K27me3, and tubulin (experiment representative of 3). (D) Karpas422 cells were subcutaneously injected in immunodeficient AGR129 mice. Mice were treated during 10 days with vehicle or 150 mg/kg GSK126 as described in “Materials and methods.” IRE1, H3K27me3, H3 total, and tubulin levels were analyzed by immunoblot. (E) Human IRE1 and DNAJB9 relative mRNA values were assessed in postmortem biopsies. (F) Parental and IRE1-deficient SUDHL4 cells were treated for 4 days with vehicle or 5 μM GSK343. IRE1, H3K27me3, H3 total, and tubulin levels were assessed by immunoblot. (G) Viability of IRE1-proficient and IRE1-deficient SUDHL4 cells was assessed by MTS/PMS assay after treatment with 10 μM GSK343 for 48 hours. P values were obtained using the unpaired Student t test; **P ≤ .01.