Western blot and qRT-PCR analysis of Mcl-1, Bcl-2, and Bcl-xl expression in MM cells during bortezomib treatment. (A) Top panel: Western blot analysis of bortezomib-treated (for 0, 6, 12, and 24 hours) human LP1 and RPMI-8226 cells and murine 5T33vt cells. The blots were probed with Mcl-1, Bcl-2, Bcl-xl, and β-actin Abs. The concentrations of bortezomib used in human LP1 and RPMI-8226 and murine 5T33vt cells were 50, 10, and 5nM, respectively. The data are representative of 3 independent experiments. Bottom panel: amounts of Mcl-1, Bcl-2, and Bcl-xl were quantified by densitometric analysis of Western blot using ImageJ Version 1.45 software. (B) The pan caspase inhibitor Z-VAD-FMK abrogates the cleavage of Mcl-1. Left panel: Western blot analysis of the Mcl-1 and caspase-3 expression in OPM2 cells. OPM2 cells were untreated (control) or were treated with 5nM bortezomib for 16 hours with or without 2 hours of pretreatment with 50μM Z-VAD-FMK as indicated. Right panel: densitometric analysis of Western blots. (C-E) qRT-PCR analysis of MCL-1, BCL-2, and BCL-XL expression at the mRNA level. The MM cell lines RPMI-8226 (10nM), MMS1 (10nM), U266 (5nM), LP1 (100nM), OPM2 (5nM), and 5T33vt (2.5nM) were treated with bortezomib for 24 hours. RNA was isolated and subjected to qRT-PCR using primers for MCL-1, BCL-2, BCL-XL, and β-actin. Beta-actin was used as an internal control. n = 3 in all experiments. *P < .05 and **P < .01 versus vehicle-treated samples. The columns show the mean results representative of 3 similar experiments; the bars indicate SD.