Figure 6.

Apoptosis induced by Bcl-X_L/Mcl-1 inhibition occurs in absence of activator BH3-only proteins. (a) Mcl-1-deficient MEFs were treated with control siRNAs or siRNAs targeting Puma, Bid, and Bim simultaneously. mRNA levels were measured 24 h after transfection. Data show means±S.D. of three independent experiments. (b) Mcl-1-deficient MEFs were transfected with control siRNAs or siRNAs targeting Puma, Bid and Bim. Forty-eight hour after transfection, cells were transfected with Bcl-X_L or control siRNAs and cell death was measured 24 h later. Data show means±S.D. of three independent experiments. (c) MEFs from Bim/Puma-double-deficient or WT mice were transfected with Bcl-X_L and Mcl-1 or control siRNAs. Cell death was measured 3 days after transfection. (d) MEFs from Bim/Puma-double-deficient mice were transfected with control or Bid-specific siRNA for 3 days. Then, cells were seeded and transfected with control, Bcl-X_L-, Mcl-1- and Bid-specific siRNAs as indicated. Bid was quantified by immunoblotting 3 days after the second transfection. Results are representative of three independent experiments. (e) Cell death was measured in Bim/Puma-double-deficient MEFs treated as described in d. The mean percentage±S.D. of Annexin V-/propidium iodide-positive cells of three independent experiments is given. (f) Upper panel: representative histogram of Bim/Puma-double-deficient MEFs treated as described in d indicating cells undergoing apoptosis as demonstrated by an Annexin V-positive/propidium iodide-negative cell population. Lower panel: quantification of caspase-3 activation by immunoblotting. Results are representative of three independent experiments. (g) Bid/Bim/Puma-triple-deficient MEFs were transfected with Bcl-X_L- and Mcl-1-specific siRNAs or control siRNAs. Cell death was assessed after 48 h. For Bcl-X_L and Mcl-1 two different siRNA sequences (siRNA a or b) were used in different combinations. Inset shows result of knockout of Bid using CRISPR/CAS9 on Bim/Puma DKO MEF to generate triple KO cells. (h) Primary human fibroblasts were treated simultaneously with Bcl-X_L-, Mcl-1- and Bak-specific siRNAs. Three days after transfection mitochondria were prepared. Mitochondria were treated with recombinant Bax (1000 nM) to stimulate cytochrome c (Cyt-c) release. Cyt-c release was assessed by immunoblotting in supernatant (SN) and mitochondrial (M) fractions. Tom40 (translocase of the outer mitochondrial membrane protein 40) served as a control for mitochondria preparation. (i) Mitochondria of Bax/Bak-double-deficient MEFs were treated with recombinant Bax. Cyt-c release was measured with 1000 nM Bax. A representative blot of two experiments is shown. *P≤0.05