Fig. 3. Dnr1 RNAi enhances caspase activation and activity by actinomycin D in S2 cells. (A) Western blot analysis of lysates from control S2 cells and S2 cells incubated with actinomycin D using an antibody that detects active caspase-3 (upper panel). Both treatments were performed in duplicate. Actin levels are shown as a loading control (lower panel). Actinomycin D treatment results in the appearance of an active caspase-3 signal by 6 hours. (B) Western blot analysis of lysates from control S2 cells (lanes 1-2) decay dsRNA-treated cells (lanes 3-4), Dredd dsRNA-treated cells (lanes 5-6), Drice dsRNA-treated cells (lanes 7-8) and Dronc dsRNA-treated cells (lanes 9-10). Where indicated, cells had been incubated with actinomycin D for 6 hours. Lysates were probed with an active-caspase 3 antibody. An active caspase-3 signal is detected in S2 cells, as well as Dredd and Decay dsRNA-treated cells, whereas depletion of Drice or Dronc abrogates the signal. A crossreacting band is indicated by an asterisk. (C) Time course of caspase activation by actinomycin D in lysates from S2 cells (first panel) and lysates from S2 cells treated with Dnr1 dsRNA (third panel). Actin levels are shown as a loading control for both samples (second and fourth panel, respectively). Dnr1 RNAi results in an earlier and stronger appearance of the active caspase isoform at all times measured. (D,E) Time courses of DEVDase (D) and YEVDase (E) activity from actinomycin-D-treated lysates from control S2 cells and Dnr1 RNAi treated S2 cells. Results are the mean of three independent experiments and error bars indicate standard errors. ^*P<0.01, values that differ significantly from untreated cells. Dnr1 depletion results in a significant increase of caspase activity in response to actinomycin D.