Blocking of different protein kinases and its effect on Mcl-1 expression and chemotherapeutic drug-induced apoptosis. (A) Huh7 cells were treated with the kinase inhibitors LY294002 (25 μM, blocking PI3 kinase), PD98059 (50 μM, blocking MEK1), SP600125 (1–100 μM, blocking JNK1), AG1478 (0.5–5 μM, blocking EGF receptor tyrosine kinase), rapamycin (50 pM, blocking mTOR kinase) and Raf I-kinase inhibitor (0.1–1 μM) for the time indicated. Whole cell lysates were prepared, separated, and immunoblotted with antibodies against Mcl-1 and alpha-tubulin. Blots shown are representative for at least two independent experiments. (B) Huh7 cells were treated with LY294002 (25 μM), PD98059 (50 μM), rapamycin (50 pM). After 1 h of pre-incubation, cells were additionally treated with epirubicin, 5-FU or left untreated for 24 h (concentrations as indicated). Cells were then harvested and analyzed by flow cytometry for apoptosis induction according to Nicoletti et al. . Assays were performed in triplicates and are representative for three independent experiments. Values are means + SD. *p < 0.01, **p < 0.001.