Skip to main content
. 2010 Nov 19;18(4):645–655. doi: 10.1038/cdd.2010.137

Figure 2.

Figure 2

UV-induced JNK activation leading to apoptosis is specifically enhanced by RASSF7 knockdown. (a and b) Increased p-JNK but unaltered p-p38. HeLa cells treated with control or RASSF7 siRNA were exposed to UV (100 J/m2) and cultured for the indicated times. Lysates were immunoblotted (top) to detect total and phosphorylated JNK (p-JNK; a), and total and phosphorylated p38 (p-p38; b). Results were quantitated by densitometry from blots and expressed as mean fold stimulation (phosphorylated/total)±S.D. over levels in control cultures not exposed to UV. *P<0.05 and **P<0.01, compared with control siRNA. (c) Increased c-Jun phosphorylation. HeLa cells treated with control or RASSF7 siRNA for 24 h were transfected with FLAG–JNK1 for 24 h. Cells were exposed to UV (100 J/m2) and cultured for 1 h. Immunoprecipitated JNK1 using anti-FLAG beads were subjected to an in vitro kinase assay using GST–c-Jun as the substrate. The original lysates, the immunoprecipitates and the assay products were immunoblotted to detect the indicated proteins. α-tubulin, loading control. Fold stimulation, densitometric evaluation of the relative increase in lanes 2–4 over lane 1. (d) Specificity for JNK. HeLa cells treated with control or RASSF7 siRNA were pretreated with DMSO (control) or 20 μM SP600125 (JNK inhibitor) or SB203580 (p38 inhibitor) for 1 h. Cells were then exposed to UV (100 J/m2) and cultured for 3 h with inhibitors. Cells positive for cleaved caspase-3 were evaluated as for Figures 1c and d. (e) Late phase effect on cleaved caspase-3. HeLa cells treated with RASSF7 siRNA were exposed to UV (100 J/m2) at time 0 and incubated for 3 h. During the incubation, SP600125 (20 μM) was also added at times indicated. Numbers of cells positive for cleaved caspase-3 were determined as for d. (f) Late phase effect on p-JNK. HeLa cells treated with RASSF7 siRNA were exposed to UV (100 J/m2) and cultured for the indicated times. SP600125 (20 μM) was added at 60 min post-UV. JNK activation was quantitated as for a. *P<0.05 and **P<0.01, compared with without SP600125