Pharmacological inhibition of GSK-3β protected HCN cells from insulin withdrawal-induced ACD. (A) The GSK-3 inhibitor BIO (0.25 μM) markedly decreased the cell death induced by insulin withdrawal. (B) BIO strongly inhibited GSK-3β activity, as shown by a restoration of the β-catenin level, and attenuated autophagic flux, as demonstrated by Western blotting analyses of the LC3-II level 24 h after insulin withdrawal. (C) Quantitative analyses of the LC3-II and β-catenin levels after normalization to β-actin, and phos-GSK-3β level after normalization to total-GSK-3β. Quantitative data are presented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01. (D) The autophagic flux rate was estimated by GFP-LC3 punta assay in HCN cells by counting more than 50 cells from three independent experiments. The representative results are shown. Scale bar is 10 μm. (E) Quantitative data are presented as the mean ± SD (n = 3). **p < 0.01. Statistical significance was determined with an ANOVA test.