Figure 2

The effect of S44563 on apoptosis activation. (a) Distinct apoptotic response to S44563 in SCLC cells lines. SCLC cells were incubated with S44563 at a dose of 100 nmol/l for 24 h and then were harvested for immunoblotting. The monoclonal mouse anti-human cleaved caspase 3 clone 100 (1 : 500 dilution; Cell signaling), was used. (b) Effect of S44563 on the dissipation of the mitochondrial transmembrane potential. H146 cells were harvested after different time exposure to S44563 at different concentrations. Cells were then washed with PBS and stained with the following probes to assess apoptosis-associated modifications: propidium iodide (2 μg/ml, Sigma-Aldrich) for viability and dihexyloxacarbocyanine iodide (DiOC₆(3), 40 nmol/l, Molecular Probes) for Δψ_m dissipation (columns, mean: bars, standard error deviation; *P<0.05 for DiOC6low fractions as compared with control). Experiments were performed in triplicate. Results from two experiments were pooled. (c) Effect of S44563 on caspase activation. H146 cells were incubated with S44563 at different doses of S44563 with or without (Z-VAD-FMK, 20 μmol/l) for 24 h and then were harvested for immunoblotting. The monoclonal mouse anti-human cleaved caspase-3 clone 100 (1 : 500 dilution; Cell signaling), cleaved and full-length caspase-9 (1 : 500 dilution, Cell signaling), and cleaved caspase- 8 (1 : 500 dilution; Cell signaling) were used. (d) Effect of S44563 on sub-G1 phase induction. Cells were harvested after 24 h exposure to S44563 at different concentrations with or without the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK, 20 μmol/l, from Bachem, Weil am Rhein, Germany). Resulting DNA distributions were analyzed by Modfit (Verity Software House, Inc., Topsham, ME, USA) for the proportion of cells in sub-G₀, G₁, and G₂-M phases of the cell cycle (columns, mean; bars, standard deviation). Experiments were performed in triplicate. Results from two experiments were combined