MKC-3946 blocks XBP1 splicing and enhances cytotoxicity induced by bortezomib or 17-AAG. (A) RPMI 8226 cells were treated with bortezomib (Bor; 10nM) or 17-AAG (AAG; 1μM), in the presence or absence of MKC-3946 (10μM) for the indicated times. Total RNA was extracted, and XBP1 and β-actin mRNA were evaluated by RT-PCR. Whole-cell lysates were subjected to Western blotting using anti-IRE1α, phospho-IRE1α (p-IRE1α), BiP /GRP78, and GAPDH Abs. (B) RPMI 8226 cells were treated with bortezomib (Bor; 10nM) in the presence or absence of MKC-3946 (10μM) for the indicated times. Nuclear extracts were subjected to Western blotting using XBP1 Abs. Lamin B served as a loading control. (C) RPMI 8226 and INA6 cells were treated with bortezomib or 17-AAG in combination with MKC-3946, 0μM (□), 5μM (▩), or 10μM (■), for 48 hours. Cell proliferation was assessed by [³H]-thymidine uptake of quadruplicate cultures, expressed as a percentage of untreated control. Data represent mean ± SD. (D) Primary MM cells isolated from 3 patients were treated with bortezomib or 17-AAG in combination with MKC-3946 0μM (□), 5μM (▩), or 10μM (■) for 36 hours. Cell viability was assessed by MTT assay of triplicate cultures, expressed as a percentage of untreated control. Data represent mean ± SD.