FIGURE 1.
siRNA-mediated STAT1 knockdown protects INS-1E and primary rat β-cells against cytokine-induced apoptosis. A–F, insulin-producing INS-1E cells were left untransfected (NT), or transfected with 30 nm si-control, siIRF-1, or siSTAT1. After 24 h of recovery, cells were left untreated or exposed to 10 units/ml IL-1β and 100 units/ml IFN-γ for 12 or 24 h as indicated. A, STAT1, IRF-1, and α-tubulin protein expression were evaluated by Western blot. B and C, mean optical density measurements of STAT1 and IRF-1 Western blots corrected for protein loading by α-tubulin. Results are mean fold variation ± S.E. of five independent experiments. D and E, apoptosis was evaluated using Hoechst 33342/propidium iodide staining (D) and a Cell Death Detection ELISAPLUS kit (E). F, nitrite concentrations in supernatants were evaluated as described under “Experimental Procedures.” Results are mean ± S.E. of five independent experiments. G and H, primary FACS-sorted rat β-cells were cultured for 2 days and then left untransfected or transfected with 30 nm of si-control, siIRF-1, or siSTAT1 as indicated. After 24 h of recovery, cells were left untreated or exposed to 10 units/ml IL-1β and 100 units/ml IFN-γ for 24 h as indicated. G, apoptosis was evaluated using Hoechst 33342/propidium iodide staining. H, nitrite concentrations in supernatants. Results are mean ± S.E. of five independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus untreated (i.e. not treated with cytokines) or untreated transfected with the same siRNA. §§, p < 0.01 and §§§, p < 0.001 versus untransfected and si-control treated with cytokines at the same time point, ANOVA followed by Student's t test with Bonferroni correction.