Figure 5.

Exogenous C3 prevents cytokine- and C3 deficiency–induced apoptosis. (a and b) C3aR1 mRNA expression was analyzed by real-time polymerase chain reaction (RT-PCR) and normalized by the housekeeping gene GAPDH or β-actin in, respectively, (a) INS-1E cells and (b) dispersed human islets. (c) INS-1E cells and (d) dispersed human islets were pretreated with the indicated C3 concentrations for 2 hours. Cells were then left untreated (NT) or treated with IL-1β plus IFN-γ in the absence or presence of exogenously added C3 (0.5 or 1.0 µg/mL) for (c) 24 hours or (d) 48 hours; apoptosis was evaluated using HO and PI staining. (e and f) INS-1E were left untreated (NT) or treated with IL-1β plus IFN-γ in the absence or presence of exogenously added C3 (1.0 µg/mL) and/or C3aR1 antagonist SB 290157 (10 μM) for 24 hours. (e) Cleaved caspase-3 and α-tubulin were measured by western blot. (f) Apoptosis was evaluated using HO and PI staining in INS-1E cells. (g–j) INS-1E cells were transfected with siCtrl (C) or siRNA targeting rat C3 (siC3 no. 1, C3). Cells were then left untreated (NT) or treated with IL-1β plus IFN-γ (10 and 100 U/mL, respectively) in the absence or presence of exogenously added C3 (1.0 µg/mL) for 16 hours. (g) C3 mRNA expression was analyzed by RT-PCR and normalized by the housekeeping gene GAPDH. (h) Apoptosis was evaluated using HO and PI staining in INS-1E cells. (i and j) Cleaved caspase-3 and α-tubulin were measured by western blot. Data represent the means ± SEM of four to seven independent experiments. *P ≤ 0.05, **P < 0.01, and ***P < 0.001 vs nontreated with cytokines (NT) and transfected with the same siRNA for (g–j); ^#P ≤ 0.05 and ^###P < 0.001 as indicated by bars; ANOVA followed by Student t test with Bonferroni correction.