Fig. 3:

Time Course of Caspase-3 activation in S2 cells. (A-C) In-Cell quantitative analysis of S2 cells treated for the indicated periods with Actinomycin D (Act D) to induce apoptosis. Each time point was measured in triplicate and stained for anti-active-caspase-3 (A) and f-actin (B). Panels A and B are false-colored and merged in (C) with active caspase-3 in green and f-actin in red. (D) Active-caspase-3 protein levels from (A) were quantified and normalized to f-actin levels in (B) for each time point. The active caspase-3:f-actin ratio at 0min Actinomycin D exposure was assigned a value of one and all other ratios are shown relative to this value. Error bars represent the standard error of each independent measurement. Exposure of S2 cells to Actinomycin D increased the relative levels of active-caspase-3 over time. (E) A stable S2 cell line that expresses Grim under control of the metallothionine promoter was incubated with CuSO₄ for the indicated periods. The relative levels of apoptosis at each time point were determined by flow cytometry. The level of apoptosis at 0min copper exposure was assigned a value of one and all other ratios are shown relative to this value. Induction of Grim expression results in a steady increase in the levels of apoptosis.