Figure 5

Over-expression of GSK-3β potentiated ACD without inducing apoptosis in insulin-deprived HCN cells. (A) An experimental scheme for the over-expression of GSK-3β constructs. CDA, cell death assay. (B) The GSK-3β WT and CA forms potentiated cell death, with more profound effects observed for the CA form in insulin-deprived HCN cells. EV, empty vector. (C) The potentiation of autophagy was well correlated with the elevated activities of GSK-3β. Alteration of the autophagy level was verified by Western blotting analyses of LC3-II 24 h after insulin withdrawal. Caspase-3 was not activated in spite of the increase in cell death in GSK-3β WT or CA-expressing cells. STS was treated for 6 h in I(+) as a positive control for caspase-3 activation. The over-expression of ectopic GSK-3β forms was confirmed by Western blotting analyses with an anti-HA antibody. (D) Quantitative analyses of the LC3-II level after normalization to β-actin. Quantitative data are presented as the mean ± SD (n = 3). ***p < 0.001. (E) The absence of apoptosis induction following GSK-3β over-activation was confirmed by Annexin-V staining and fluorescence-activated cell sorting analysis. Regardless of GSK-3β over-expression, the low percentages of Annexin-V-positive cells remained unaltered 24 h after insulin withdrawal. STS treatment for 6 h in I(+) was used to induce apoptosis as a positive control for Annexin-V staining and subsequent flow cytometry analysis. (F) Knockdown of Atg7 attenuated cell death. HCN cells were infected with pLKO.1- Atg7 shRNA and control scramble shRNA lentivirus. HCN cells with reduced level of Atg7 were transfected with GSK-3β CA form and cell death was measured 24 h after insulin withdrawal. Quantitative data are presented as the mean ± SD (n = 3). *p < 0.05. (G) Atg7 level and expression of HA-tagged GSK-3β CA were analyzed by Western blot analysis. STS, staurosporine.