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. 2012 Dec 20;3(12):e447. doi: 10.1038/cddis.2012.185

Figure 3.

Figure 3

Bim and Bid are crucially involved in the synergistic induction of endothelial cell death by paracetamol plus TRAIL. skHep-1 or EA.hy926 cells were treated for 6 h with 10 or 20 mM paracetamol, and 30 ng/ml, respectively, 3 ng/ml TRAIL, or the combination thereof, and Bid, Bim and tubulin as loading control were detected by western blot (a). bim-mRNA expression was measured by quantitative RT-PCR in both cell lines. GAPDH was used to normalize bim-expression levels (b). skHep-1 or EA.hy926 cells were transfected with control (siNone), Bim- or Bid-specific siRNA or the combination of both. Apoptosis sensitivity to paracetamol (10 mM), TRAIL (30, respectively 3 ng/ml) or both was analyzed by Annexin V staining after 16 h (c). Cytoplasmic (C) and mitochondrial fractions (M) were isolated. Cytochrome c (Cyto c), porin and, for comparison, twere analyzed in the different fractions by western blot (d). Mean values±S.D. triplicates are shown for quantitative RT-PCR. A typical experiment out of five is shown for western blot analysis. bim-mRNA expression, siRNA downregulation as well as cytochrome c western blot were repeated three times yielding similar results. *P<0.05, **P<0.005, ***P<0.001