Figure 7

RNAi targeting of BIM expression protects MCF-7 cells from apoptotic cell death induced by 4-OHT, MIF, and/or U0126 treatments. (a, c) BIM expression was reduced in all treatment groups by RNAi targeting. MCF-7 cells were seeded in DMEM/F12 medium containing 5% DCC FBS and, after 24 hours, transfected with scrambled or BIM-targeting RNAi. Forty-eight hours after transfection, the cells were treated with E2, E2 + 4-OHT, or E2 + 4-OHT + MIF in the absence (a) or presence of IGF-1 (20 ng/ml) (c, d) and/or U0126 (5 mM). Cells were harvested for protein at 48 hours (a, c) or 72 hours (d) after treatment and subjected to immunoblot analysis to determine levels of Bim, cleaved PARP, and cleaved lamin (a) β-actin levels served as a loading control. (b) BIM knockdown via siRNA targeting reduces the levels of ROS generated by 1 hour of treatment with 4-OHT and/or MIF. Values are expressed as mean ± SD. Statistical significance was identified between the following treatment groups: (a) scrambled E2 + 4-OHT versus siBimE2 +4-OHT; (b) scrambled E2 + MIF versus siBim E2 + MIF; (c) scrambled E2 + 4-OHT + MIF versus siBim E2 + 4-OHT + MIF; (d) scrambled E2 + 4-OHT + U0126 versus siBim E2 + 4-OHT + U0126; (e) scrambled E2 + MIF + U0126 versus siBim E2 + MIF + U0126; and (f) scrambled E2 + 4-OHT + MIF + U0126 versus siBim E2 + 4-OHT + MIF + U0126. *P < 0.001.