Figure 4

SHH signaling regulates apoptosis by COP1-mediated p53 degradation. (A-D) A549 cells were either pretreated with SHH signaling specific inhibitors like Cyclopamine or Betulinic Acid followed by infection with BCG and treatment with TNF-α (A) or transfected with SHH siRNA followed by BCG and TNF-α treatment (B) or transfected with SHH overexpression construct followed by TNF-α treatment (C and D). Analysis of apoptosis in A549 cells determined using Annexin V staining with MFI quantitation (A-C) and by quantitative real time RT-PCR for pro-apoptotic gene expressions (D). *p < 0.05 (one-way ANOVA), as compared to TNF-α treated cells (A); or compared to pcDNA3 transfected, TNF-α treated cells (C); **p < 0.005 (one-way ANOVA), as compared to BCG + TNF-α treated cells and ns, not significant, as compared to TNF-α treated SHH siRNA-transfected cells. (E) A549 cells were infected with BCG for 12 h prior to TNF-α treatment. Cells were then treated with MG132 (20 μM) for 4 h before harvest as indicated. Complexes immunoprecipitated with anti-p53, anti-UbK48 or anti-COP1 were analyzed by immunoblotting with indicated antibodies. Data is representative of mean ± SEM of at least 3 different experiments and all blots are representative of 3 independent experiments. Med, Medium; NT, non-targeting. Bar, 20 μm.