GSK-3β activation accelerated autophagic flux in insulin-deprived HCN cells. (A) An experimental scheme for the assessment of autophagic flux. (B) The autophagic flux rate was estimated by Western blotting analyses in GSK-3β CA-expressing HCN cells 6 h after insulin withdrawal. Bafilomycin A1 (BafA, 30 nM) was added 2 h before cell harvest. (C) The autophagic flux rate was estimated by GFP-LC3 punta assay in GSK-3β CA-expressing HCN cells 6 h after insulin withdrawal. BafA (30 nM) was added 2 h before imaging. The results shown are representative of three independent experiments. (D) Quantitation of GFP-LC3 puncta. More than 120 cells per condition were counted from three independent experiments. Quantitative data are presented as the mean ± SD (n = 3). Scale bar is 10 μm. *p < 0.05, ***p < 0.001. Statistical significance was determined with an ANOVA test. EV, empty vector, BafA, Bafilomycin A1.