PDF file - 4200K, Supplementary Figure 1 (A) MM.1R and RPMI-8226 MM cells were treated with mel-flufen {1μM and 3μM, respectively} for 24h; protein lysates were subjected to immunoblotting using indicated antibodies. (B) MM1.R cells were treated with increasing concentrations of mel-flufen for 16h; cells were then stained with DCF-DA (2'-7'-Dichloro-Fluorescein Diacetate) and ROS production was assessed by flow cytometry (mean � SD; P < 0.05, n=3). As a positive control, bortezomib-treated cells were also assessed for ROS generation. (C) MM.1R cells were treated with indicated concentrations of mel-flufen for 16h; cells were then stained with the cationic dye, JC-1 and the disruption of mitochondrial transmembrane potential was assessed using by flow cytometry (mean � SD; P < 0.05, n=3). (D) RPMI-8266, MM.1R and MM.1S cell were treated with mel-flufen (3 μM, 1 μM, and 1 μM, respectively) for 24h and cytosolic extracts were analyzed for cytochrome-c release by ELISA (n = 3, mean � SD; P < 0.05). (E) MM.1S, MM.1R, and RPMI-8226 cells were treated with mel-flufen in the presence or absence of ZVAD-FMK for 24h, and cell viability was measured using MTT assay (n = 3, mean � SD; P < 0.001).