Figure 6

Treatment of BT474 cells with the mTORC1 inhibitor RAD001 decreases Myc and Bim expression and mitigates Mcl-1 dependence. (A-B) BT474 cells were treated with 20 nM RAD001 for 72 hours. (A) Western blot analysis was performed to document the effect of RAD001 treatment on Mcl-1, c-Myc and Bim expression. (B) Cell death was assessed by Apo 2.7 staining as described in Materials and Methods. Data are mean ± se of three independent experiments. p-values were assessed using a Student's t test. (C) BT474 cells were treated with 20 nM RAD001 or left untreated, as indicated. 24 hours later, cells were transfected with control or Mcl-1 siRNA and put back into control or RAD001 containing complete medium. Apoptosis was assessed 48 hours later by Apo 2.7 staining as described in Figure 1. Data are mean ± se of the indicated number of independent experiments. p-values were assessed using a Student's t test. (D) Western blot analysis was performed to document the effects of RAD001 treatment and of Mcl-1 siRNA on the phosphorylation of p70S6K and on Mcl-1, Bim and XIAP expression, using ß-tubulin as a loading control.