Fig. 6: GSK3β knock-down is sufficient to decrease GR expression and GC-induced β-cell death.

A Representative Western Blot (left) and quantification (right) of GSK3β and GSK3α levels in INS-1 832/13 after 24 h incubation with GSK3β siRNA (upper panel, n = 6) or with GSK3α siRNA (lower panel, n = 6). β-actin is used as loading control for GSK3α and tubulin is used as loading control for GSK3β. Results are expressed as percentage of siCTL condition, represented by dashed line, for each independent experiment. B Representative Western Blot (left) and quantification (right) of GR protein level in INS-1 832/13 after 24 h incubation with GSK3β siRNA (upper panel, n = 6), or with GSK3α siRNA (lower panel, n = 6). Tubulin is used as loading control. Results are expressed as percentage of siCTL condition for each independent experiment. C Relative mRNA level of GSK3β (upper panel) and GSK3α (lower panel), measured by qPCR, in INS-1 832/13, after infection with lentivirus coding for scrambled shRNA (shScr), GSK3β.1 shRNA (shGSK3β.1) or GSK3β.2 shRNA (shGSK3β.2) (n = 3). Results are expressed as percentage of scrambled shRNA condition, represented by dashed line, for each independent experiment. D Representative Western-blot (upper panel) and quantification (lower panel) of GR protein level in GFP sorted INS-1 832/13 after infection with lentivirus coding for scrambled shRNA or GSK3β.2 shRNA (n = 2). β-actin is used as loading control. Results are expressed as percentage of scrambled shRNA condition for each independent experiment. E Cell death reported by 7-AAD incorporation measured by flow cytometry, and quantification (n = 5), after infection of INS-1 832/13 cells with lentivirus coding for scrambled shRNA, GSK3β.1 shRNA or GSK3β.2 shRNA, and 24 h after incubation with (right panel) or without Dexamethasone (Dexa) (left panel). Cyclophilin A is used as housekeeping gene in figure C. Results are shown as means ± S.E.M. n represents the number of independent experiments. *p < 0.05; **p < 0.01.