C3 inhibition exacerbates chemokine expression and release. (a–d) INS-1E cells were transfected with siCtrl or siRNA targeting rat C3 (siC3 no. 1). Cells were then left untreated or treated with IL-1β plus IFN-γ (10 and 100 U/mL, respectively) as indicated under the figure. CCL2 (a), CCL5 (b), and CXCL10 (c) mRNA expression was analyzed by real-time polymerase chain reaction (RT-PCR) and normalized by the housekeeping gene GAPDH. (d and e) CXCL10 release was measured by ELISA in (d) INS-1E cells and (e) dispersed human islets. (e) Dispersed human islets were transfected with siCtrl or siRNA targeting human C3 (siC3h). Cells were then left untreated or treated with IL-1β plus IFN-γ (50 and 1000 U/mL, respectively) for 48 hours. In (d), CXCL10 release was normalized by protein content, whereas CXCL10 release in (e) was normalized by number of cells. Results are means ± SEM of three to six independent experiments. *P ≤ 0.05, **P < 0.01, and ***P < 0.001 vs transfected with (a–d) siCtrl vs (e) nontreated with cytokines (NT) and transfected with the same siRNA; #P ≤ 0.05, ###P < 0.001 as indicated by bars; ANOVA followed by Student t test with Bonferroni correction.