Fig. 3. Stimulation of S. cerevisiae acetic-acid-induced apoptosis by expression of mammalian PKC-, -, - or - is not mediated by yeast caspase activation. (A) Flow cytometric analysis. Percentage of cells with active caspases obtained with yeast transformed with the empty vector (YEplac181 or YEp51) and with yeast expressing PKC-, -, - or -, after 1 hour treatment with 400 mM acetic acid. Treated cells were labelled with 50 µM FITC-VAD-FMK for 25 minutes at 30°C and analysed by flow cytometry. Cells treated in the absence of acetic acid (0 mM) were used as negative control. Data represent one of two independent experiments. (B) Effect of the caspase inhibitor Z-VAD-FMK on the survival of yeast expressing PKC- treated with 400 mM acetic acid. Before treatment with acetic acid, cells were pre-treated without (control) or with 100 µM Z-VAD-FMK for 2 hours at 30°C. The percentage of dead cells was determined by c.f.u. counts, as in Fig. 1. Data are the mean ± s.e.m. of two independent experiments with six replicates. Significant differences from those obtained with the empty vector are indicated by ^*P<0.05 (unpaired Student's t-test). (C) Fluorimetric analysis. Cell extracts obtained from yeast expressing PKC- and treated with 0 mM (negative control) or 400 mM acetic acid, were incubated with 50 µM of the fluorogenic caspase substrates Ac-DEVD-AMC, Ac-IETD-AMC or Ac-VEID-AMC. Recombinant caspase 3 (for Ac-DEVD-AMC), caspase 6 (for Ac-VEID-AMC) and caspase 8 (for Ac-IETD-AMC) were used as positive control. Aminomethylcoumarin (AMC) release was monitored for 90 minutes at 30°C in a spectrofluorimeter. Caspase activity is expressed in arbitrary fluorescence units (FU)/minute. Data are the mean ± s.e.m. of two independent experiments.