Dominant Negative FoxO1 in Insulin-Secreting Cells

MIN6 and HEK293 cells were purchased from AddexBio Technologies and ATCC, respectively. MIN6 cells were maintained in DMEM with 4.5 g/L glucose and L-glutamine without sodium pyruvate (Corning) containing 15% fetal bovine serum (FBS), penicillin-streptomycin and 0.05 mM 2-mercaptoethanol. For gene expression analysis, MIN6 cells were transduced with adenovirus encoding dominant negative FoxO1 and GFP at an MOI of 500 and incubated for 48 h. Cells were also treated with FoxO1 inhibitor (AS18420856, 100 nM) and incubated for 24 h. HEK293 cells were maintained in DMEM with 1.0 g/L glucose and L-glutamine without sodium pyruvate (Corning) containing 10% FBS and penicillin-streptomycin. Islets were isolated by collagenase digestion as previously described [17 (link)]. Isolated islets were incubated overnight in RPMI 1640 containing 10% FBS with penicillin-streptomycin and used for further analyses.