Knockdown of GSK-3β protected HCN cells from insulin withdrawal-induced ACD. (A) An experimental scheme for the knockdown of GSK-3 isoforms. CDA, cell death assay. (B-C) GSK-3β-targeting siRNA (30 nM) substantially decreased cell death (B), and attenuated autophagy after insulin withdrawal (C). Knockdown of GSK-3α barely affected ACD. Scrambled siRNA (30 nM) was added to GSK-3α or β-specific siRNA to keep the same total siRNA concentrations at 60 nM. Quantitative data are presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01. (D) Quantitative analyses of the LC3-II and β-catenin levels after normalization to β-actin. Quantitative data are presented as the mean ± SD (n = 3). *p < 0.05. Statistical significance was determined with an ANOVA test.