Bortezomib activates the UPR in MM cells. (A-B) qRT-PCR analysis of the ER stress markers GRP-78 and CHOP expression at the mRNA level. The MM cell lines RPMI-8226 (10nM), MMS1 (10nM), LP1 (100nM), OPM2 (5nM), and 5T33vt (2.5nM) were treated with bortezomib for 24 hours. n = 3 for all experiments. *P < .05 and **P < .01 versus vehicle-treated samples. (C) Left panel: Western blot analysis of GRP-78 and CHOP expression. Whole-cell extracts were prepared from bortezomib-treated (for 0, 6, 12, and 24 hours) human LP1 and MMS1 cells and murine 5T33vt cells; the concentrations of bortezomib used were 100, 10, and 5nM, respectively. The data are representative of 3 independent experiments. Right panel: amounts of GRP-78 and CHOP were quantified by densitometric analysis of Western blot using ImageJ Version 1.45 software. (D) Western blot analysis of the UPR arm of ATF4 activation in bortezomib-treated MM cells. The doses of bortezomib were used at 10nM in both RPMI-8226 and MMS1 cells and at 2.5nM in 5T33vt cells. Left panels show the representative blots of 3 independent experiments; right panels show densitometric analysis. (E-F) qRT-PCR analysis of ATF4 and its target gene ATF3 expression in bortezomib-treated MM cells. The MM cell lines RPMI-8226 (10nM), MMS1 (10nM), LP1 (100nM), OPM2 (5nM), and 5T33vt (2.5nM) were treated with bortezomib for 24 hours. Data represent the means ± SD for 3 separate experiments. *P < .05 and ** P < .01 versus vehicle-treated samples.