GCB DLBCLs display impairment in IRE1 expression that affects the ER stress response. (A-B) Representative ABC DLBCL and GCB DLBCL cell lines were treated with 5 μg/mL tunicamycin (TM) for 6 hours and analyzed by quantitative real-time PCR for expression of XBP1 target genes Sec23b, DNAJB9, and SRPR mRNA levels relative to β-actin (mean and standard deviation [SD] of technical triplicates of 1 representative experiment of 3) (A). XBP1 mRNA splicing was measured by RT-PCR analysis of XBP1 mRNA maturation (B) DNA electrophoresis is shown in the top panel. Protein levels of XBP1s and IRE1 protein were analyzed by immunoblot. Tubulin (Tbl) is used as loading control (B, below). (C) DLBCL cell lines were treated for 6 hours with 5 μg/mL tunicamycin (TM) or vehicle and analyzed by immunoblot for XBP1s, IRE1, ATF4, PERK, ATF6, and tubulin (Tbl). (D) Representative ABC and GCB cell lines were treated with different ER stress inducers, tunicamycin (TM), thapsigargin (Tpg), DTT, brefeldin A (Bref A), and bortezomib (Btz) for 6 hours. IRE1, XBP1s, PERK, ATF4, and Tubulin (Tbl) expression was analyzed by immunoblot. (E) Viability of IRE1-high and IRE1-low DLBCL cells assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTS)/1-methoxy phenazine methosulfate (PMS) assay after treatment of the indicated time course with 0.05 μg/mL tunicamycin or 12.5 μM nelfinavir. P values were calculated using 2-way analysis of variance (ANOVA); *P ≤ .05; ***P ≤ .001.