Expression of pro-apoptotic Bim is required for induction of cell death and decrease in mammosphere formation by Mcl-1 knock down. BT474 cells were transfected with control siRNA, Bim siRNA, PUMA siRNA and/or Mcl-1 siRNA as indicated. (A) Western blot analysis was performed to confirm the down regulation of the targeted proteins by each of the indicated siRNA. (B) Apoptosis was assessed 48 hours later by Apo 2.7 staining as described in Figure 1. Data are mean ± se of three independent experiments. They are expressed as a percentage of cell death obtained after Mcl-1 siRNA transfection of cells that had been transfected with control siRNA (46% ± 5.2%). p-values were assessed using a Student's t test. (C) Mammosphere formation by BT474 cells transfected with the indicated siRNA was evaluated as described in Figure 2. Data are mean ± se of 3 independent experiments normalized to data obtained with transfection with a control siRNA.