Figure 5
Knockdown of protein kinase Cδ (PKCδ) with siRNA and overexpression of the PKCδ proteolytic cleavage-resistant mutant (PKCδ D327A -CRM) protects against tumor necrosis factor (TNF) toxicity. (A) DNA fragmentation assay in siRNA transfected cells. N27 cells transfected with either siRNA to PKCδ or scrambled control siRNA were treated with TNF for 16 h and processed for the DNA fragmentation ELISA. Raw values were normalized to protein concentrations and expressed as the fold change over the respective controls. TNF-induced DNA fragmentation was significantly reduced in PKCδ siRNA transfected cells. (B) Suppression of the PKCδ protein levels by siRNA was confirmed by Western blotting. (C) DNA fragmentation assay in N27 cells overexpressing the cleavage-resistant PKCδ mutant protein (PKCδD327A-CRM). N27 cells stably expressing the cleavage-resistant PKCδ mutant or the β-galactosidase (LacZ) control gene were treated with TNF for 16 h and processed for the DNA fragmentation assay. Expression of the mutant PKCδ protein was confirmed by imaging the V5 tag by immunocytochemistry. (D) DNA fragmentation induced by TNF was attenuated in N27 cells expressing the caspase-3 cleavage-resistant mutant protein, indicating the proteolytic cleavage is a necessary event for TNF toxicity in these cells. Data represent the group mean ± SEM, n = 6 to 8 per group and experiments were repeated three times. *** (P <0.001) indicates significant differences with TNF treatment compared to controls in scramble siRNA and LacZ expressing cells; ## (P <0.01) indicates significant differences between the two TNF treatment groups.