Fig. 5. Overexpression of Dnr1 blocks caspase activity and apoptosis in S2 cells. (A) Anti-HA western blot analysis of lysates from control S2 cells, S2 cells expressing HADnr1 and S2 cells expressing HADnr1C563Y treated with actinomycin D as indicated. Equal amount of lysate was loaded in each lane. The levels of HADnr1C563Y are considerably higher than HADnr1, consistent with a role for the RING domain in regulating Dnr1 stability. (B) Caspase activity assays in lysates from S2 cell, S2 cells treated with actinomycin D, HADnr1-expressing cells treated with actinomycin D and HADnr1C563Y-expressing cells treated with actinomycin D. HADnr1 expression blocks actinomycin-D-induced caspase activity, whereas the RING-domain-inactive version fails to impact on caspase activity. (C) Quantification of the apoptotic index of S2 cells, HADnr1-expressing S2 cells and HADnr1C563Y expressing S2 cells before (columns 1-3) or after exposure to actinomycin D (columns 4-6). Results are the mean of three independent experiments and error bars indicate standard errors. (D) Quantification of the apoptotic index of S2 cells, HADnr1-expressing S2 cells and HADnr1C563Y-expressing S2 cells before (columns 1-3) or after exposure to ultra-violet irradiation (columns 4-6) or cycloheximide (columns 7-9). Results are the mean of three independent experiments and error bars indicate standard errors.