Mcl-1 silencing sensitizes HeLa cells to proteasome inhibitor cytotoxicity. (A) HeLa cells were treated for 4 hours with epoxomicin (0.4 μM) or MG132 (0.5 μM), or were left unstimulated. Susequently, cells were used to prepare cell lysates and Mcl-1 and γ-tubulin levels were determined by immunoblotting. (B) HeLa cells were treated for 24 hours with epoxomicin (0.4 μM), MG132 (0.5 μM), or etoposide (25 μg/mL). Subsequently, cells were harvested, stained with propidium iodide, and analyzed by flow cytometry. Results are presented as means of triplicates with SD. (C) HeLa cells were transfected with pLL3.7 plasmid or its derivatives for Mcl-1 silencing (Mcl-1-)sh1, (Mcl-1-)sh2, (Mcl-1-)sh3. At 24 hours after transfection, cells were exposed to epoxomicin (0.4 μM; ▦) or MG132 (0.5 μM; □) or not (▪). Cells were harvested 12 hours later, stained with propidium iodide, and analyzed by flow cytometry. Experiments were performed in triplicate and data are presented as means of 2 separate experiments with SD. (D) HeLa cells were transfected with pLL3.7 plasmid or its Mcl-1-sh1 derivative. At 24 hours after transfection, cells were treated with 25 μg/mL etoposide (□) or not (▪). Cells were harvested 12 hours later and analyzed by flow cytometry after staining with propidium iodide. Data are presented as means of triplicates with SD. ^*P < .05.