Figure 3

ERK1/2 signaling regulates the Mcl-1 expression. (A) Immunoblot analysis of Akt, phospho-Akt, ERK1/2 and phospho-ERK1/2 in CaOV3 and OVCAR3 cells following the addition of 10% OVC508 ascites. (B) Expression levels of Akt, phospho-Akt and Mcl-1 in vehicle, Akt or control siRNA-transfected CaOV3 and OVCAR3 cells 24 h post-transfection in the presence or absence of OVC508 ascites. (C) Immunoblot analysis of ERK1/2, phospho-ERK1/2 and Mcl-1 in OVC508 ascites- and U0126-treated OVCAR3 and CaOV3 cells 24 h post treatement. (D) Real-time PCR analysis of Mcl-1 transcript levels in CaOV3 and OVCAR3 cells. Cells were treated with either ascites (OVC508), MEK1/2 inhibitor U0126 or Akt inhibitor LY294002 and RNA was extracted 4 h later. Results were standardized using primers of the housekeeping gene RPLPO. Results are expressed as fold change relative to basal levels observed in cells incubated in the absence of ascites. (E) Apoptosis was assessed 24 h following incubation with U0126 in the presence or absence of TRAIL 50 ng/ml for CaOV3 cells and 100 ng/ml for OVCAR3 cells. Data shown are means ± SEM derived from three independent experiments. * indicates P < 0.001, ** P < 0.0001.