Mcl-1 silencing increases apoptosis in response to proteasome inhibitors. (A) HeLa cells were transfected with the Mcl-1-silencing construct (Mcl-1-sh1; □) or the control plasmid pLL3.7 (▪). At 24 hours after transfection, cells were incubated for 12 hours with or without 0.25 μM MG132. Thereafter, cells were harvested, stained with PE-conjugated annexin V, and analyzed by flow cytometry. The rate of annexin V-positive cells was determined among the fraction of EGFP⁺ cells. Data are presented as means of triplicates with SD. ^*P < .05. (B) At 24 hours after transfection with the Mcl-1-sh1-delivering plasmid or the pLL3.7 control vector, HeLa cells were exposed for 12 hours to the indicated concentrations of bortezomib. Thereafter, cells were harvested and the rate of annexin V-positive cells among the transfected cells (EGFP⁺) was determined by flow cytometry. Means of triplicates with SD are shown. Statistical analysis compares transfection with pLL3.7 (♦) versus Mcl-1-silencing plasmid (▪) on each bortezomib concentration.