Additional file 1.

α-amanitin and α-amanitin plus 5-fluorouracil can provoke a p53-mediated, transactivation-independent apoptosis in HCT116 cells. (A) Exponentially growing cultures of HCT116 and HCT116 p53-/- cells were mock-treated (m) or exposed to α-amanitin (10 μM; A), 5FU (375 μM; F), or both combined (FA). At the indicated time points, the percentage of apoptotic cells was determined by measuring the numbers of cells with a sub-2n DNA content after PI staining, as detailed in Materials and methods. (B) RT-PCR were performed on RNA from HCT116 cells after 24 h of drug treatment, as indicated, and the relative levels of the p53-responsive p21 and the control gapdh transcripts were determined. Western blot analysis confirmed the lack of stimulation of p21 under conditions of transactivation repression by α-amanitin. p53 was detected with DO-1 at a dilution of 1:2000; loading control β-actin was detected with anti-β-actin antibody diluted at 1:5000, and p21 was detected with anti-p21 antibody diluted at 1:1000. (C) Induction of apoptosis in HCT116 cells by 5FU and α-amanitin plus 5FU is mediated by cytochrome c (cyt.c) release and caspase 3 (casp3) activation. 15 μg of cytoplasmic protein (for cyt.c) or 30 μg of total protein (for casp3) from cells treated in the indicated way for 48 h were subjected to immunoblot analysis. Anti-cytochrome c antibody and anti-caspase 3 antibody (detecting both the pro-caspase and the activated caspase) were diluted at 1:1000.

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Heyne et al. Molecular Cancer 2008 7:54   doi:10.1186/1476-4598-7-54