Silencing of Mcl-1 expression in HCC cells by RNA interference results in sensitization towards chemotherapy. (A) Huh7 cells were transfected with siRNA specific for Mcl-1 (40 nM) or transfected with siRNA specific for green fluorescent protein (GFP) as control. Cells were lysed 24, 48 and 72 h after transfection and analyzed for Mcl-1 expression by Western Blot (upper panel). Tubulin expression was used to control equal loading. siRNA concentration was titrated as indicated and expression of Mcl-1 and tubulin was analyzed by Western Blot 24 h after transfection (middle panel). 24 h after transfection with siRNA (40 nM), total RNA was extracted and analyzed for Mcl-1 mRNA expression by quantitative real-time PCR (lower panel). Expression of Mcl-1 was normalized to actin in each sample. Relative Mcl-1 expression was calculated as described in the materials and methods section. (B) Huh7 cells were transfected with siRNA specific for Mcl-1 (40 nM, "Mcl-1 siRNA +") or transfected with siRNA specific for GFP as control ("Mcl-1 siRNA -"). 24 h after transfection cells were treated with different chemotherapeutic drugs for 24 h (mitomycin C, 10 μM, 5-FU, 150 μg/ml (with or without valproic acid, VA, 100 μg/ml) and epirubicin, 1 μg/ml). Some cultures were irradiated with UV (7.5 and 15 mJ/cm2, respectively). 24 h later, cells were harvested and analyzed by flow cytometry for apoptosis induction according to the method by Nicoletti . Assays were performed in triplicates and are representative for at least three independent experiments. Values are means + SD. *p < 0.01, **p < 0.001. (C) Cells were incubated for 24 h with epirubicin (1 μg/ml). Caspase-3 (upper panel) and caspase-9 (lower panel) activity were measured by fluorometric assays as described in the materials and methods section. Bars = SD.