The role of XBP1 in UPR-mediated Mcl-1 expression. (A-F) RT-PCR analysis of XBP1 splicing in different MM cell lines. The MM cell lines OPM2, MMS1, U266, 5T33vt, RPMI-8226, and LP1 were treated with bortezomib and tunicamycin for 24 hours at varying doses. RNA isolation and RT-PCR were performed as described in “Methods.” In the human MM cell lines OPM2, MMS1, U266, RPMI-8226, and LP1, the 210- and 184-bp DNA fragments correspond to unspliced and spliced human XBP1 mRNAs, respectively. In 5T33vt cells, the 343- and 327-bp DNA fragments correspond to unspliced and spliced mouse Xbp1 mRNAs, respectively. (G) Western blot analysis of XBP1 splicing in bortezomib-treated RPMI-8226 and MMS1 cells. Total protein extracts from RPMI-8226 and MMS1 cells treated with 10nM of bortezomib for 0, 6, 12, and 24 hours were electrophoresed and subjected to Western blot analysis using XBP1 Abs. Left panels show the representative blots of 3 independent experiments; right panels show densitometric analysis. (H) qRT-PCR measurement of MCL-1 and UPR-related downstream genes in U266 cells with XBP1 knockdown. n = 3 for all experiments. *P < .05 and **P < .01 versus siRNA mock samples. Data represent the means ± SD.