Fig. 2.

EDD downregulation decreases Mcl-1 protein levels, whereas Mcl-1 overexpression inhibits apoptosis upon EDD knockdown. (A) Cells were either untreated (none) or transfected with control or EDD siRNA1 for 24h. Lysates from floating and adherent cells were immunoblotted for EDD, PARP and Bcl2 family members as indicated. (B) A2780ip2 and ES-2 cells were untreated (none) or transfected with control siRNA or EDD siRNA1 and simultaneously treated with either Q-VD-OPH pan caspase inhibitor (+) or the negative control Z-FA-FMK (−). After 24h, floating and adherent cells were collected, lysed, run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted for EDD, PARP, Mcl-1, Bcl-xL, p53 upregulated modulator of apoptosis and actin. (C) A2780ip2 and (D) ES-2 cells were either untreated (none) or were transfected with the control siRNA, EDD siRNA1 or siRNA to Mcl-1 or Bcl-xL for 24h. Floating and adherent cells were fixed, stained with propidium iodide and the percentage of sub-2n cells determined by flow cytometry. P values represent significance compared with the control siRNA-transfected cells. (E) Cells were transfected with siRNA as in (D) for 24h. Lysates from floating and adherent cells were immunoblotted to confirm knockdown and to determine PARP cleavage. (F) Stable populations of ES-2 cells and (G) stable clones of A2780ip2 cells expressing either pBabe vector (Vec) or pBabe-Flag-Mcl-1 (Mcl-1) were generated by retroviral transduction. Cell lysates were immunoblotted as indicated. Arrows indicate endogenous Mcl-1 and the slower-migrating Flag-Mcl-1. (H) Stable ES-2 or (I) A2780ip2 cells were transfected with control or EDD siRNA1 for 24h and cell lysates immunoblotted as indicated. Arrows indicate endogenous Mcl-1 and Flag-Mcl-1.