A. Stable knockdowns of ATF6, IRE1α and XBP-1 were generated in Mel-RM and MM200 cells by retroviral insertion of shRNA. Transduced cells were selected with 5µg/ml puromycin. Knockdown of ATF6, IRE1α and XBP-1 was confirmed by real time-PCR.
B. Following treatment of knockdown cells with Obatoclax (1µM) or ABT-737 (10µM) for 24 hours, measurement of apoptosis was performed by the propidium iodide method using flow cytometry. Columns, mean of three individual experiments; bars, SE.
C. The stable knockdown cells were treated with Obatoclax (1µM) or ABT-737 (10µM) for indicated periods. Whole cell lysate was then subjected to Western blot analysis of Mcl-1 protein expression levels. Data are representative of three individual experiments.