Effects of PKC412 on neoplastic human MCs transfected with a Bim-specific siRNA. (A-B) Top panel: Western blot analysis of expression of Bim in HMC-1.1 cells (A) and HMC-1.2 cells (B) cultured in control medium (control) or PKC412 (1μM) for 24 hours. PKC412 was applied on nontransfected cells, on HMC-1 cells transfected with a control siRNA against luciferase (luc-siRNA), and on HMC-1 cells transfected with a Bim-specific siRNA (bim-siRNA). Western blotting was performed using an antibody against Bim and an antibody against β-actin. (A-B) Bottom panel: Evaluation of effects of PKC412 (1μM, 24 hours) on apoptosis in HMC-1.1 cells (A) and HMC-1.2 cells (B). Results show percentages of apoptotic cells and are expressed as mean ± SD of 3 independent experiments (*P < .05). (C) Annexin V stain of HMC-1 cells after transfection with a control siRNA against Luciferase (Luc; top panels) or a Bim-specific siRNA (bottom panels) and exposure to control medium (left panels) or PKC412 (1μM; right panels) for 24 hours. The percentage of apoptotic cells is also shown. (D) HMC-1.2 cells were transfected with 200nM control siRNA (Luciferase siRNA) or with 200nM siRNA directed against Bim as described in “Methods.” Thereafter, cells were split and kept in control medium or in the presence of PKC412 (1μM) or bortezomib (10nM) for 24 hours. After incubation, cells were spun on cytospin slides and stained by Wright-Giemsa. The percentage of apoptotic cells was determined by light microscopy. Results represent the mean ± SD of 3 independent experiments. *P < .05 compared with Luciferase siRNA.