Fig 1: c-Myc expression in BT474 cells impacts on Bim expression and Mcl-1 dependence. BT474 cells were transfected with control siRNA, Foxo3A siRNA or c-Myc siRNA, as indicated. 48 hours later, western blot analysis was performed to confirm the effect of each siRNA on the expression of the targeted proteins and on Bim (EL), (L-) and (S), as indicated (A-B). In (C), BT474 cells were transfected with control siRNA, c-Myc siRNA, and/or Mcl-1 siRNA as indicated and apoptosis was assessed 48 hours later 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 (42.4% ± 6.8%). p-values were assessed using a Student's t test.
Fig 2: 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.
Fig 3: Noxa induction triggered GSIXII-induced apoptosis. (A) Breast cancer cells were first transfected by siRNA targeting Bim, Puma, or Noxa, or control siRNA (siCt), and then treated or not with GSIXII for 48 hours and analyzed with flow cytometry after Apo2.7 immunostaining. Represented data are the means of positive cells ± SEM, from three independent experiments. SiRNA effects were compared with corresponding controls. (B) Expression of Noxa protein was assessed with immunoblot after 48 hours of treatment of GSIXII, 15 µM, in indicated breast cancer cell lines. (C) Noxa mRNA is induced by a GSIXII treatment. Quantitative PCR was performed on cell lines after 48 hours of treatment with mock (white) or GSIXII (black) and quantified as arbitrary units (au) compared with the mock-treated condition. Represented data are the means of positive cells ± SEM, from three independent experiments.
Fig 4: HER2-amplified mammary tumors express detectable levels of Bim. Tumor lysates were prepared and analyzed by western blotting as described in Materials and Methods.
Fig 5: Myc associates with the Bim promoter by a RAD001 dependent process in BT474 cells. (A) Schematic representation of the potential Myc binding sites of the BCL2L11 promoter. (B) BT474 cells were treated (2, 4, 6) or not (1, 3, 5) with RAD001, soluble chromatin was immunoprecipitated with anti-Myc (3, 4) or anti-E2F-1 antibodies (5, 6) and DNA samples were then amplified using primers that cover the -61/+50 region of the BCL2L11 promoter. IgG immunoprecipitations were used as controls (1, 2). Data are mean ± sd of 4 independent experiments normalized to data obtained with control IgG immunoprecipitations. p-values were assessed using a Student's t test.
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