Hek293

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.

The effect of compounds (Ar1-Ar24) on human Ca²⁺ channels was evaluated using Fluo-4 Direct calcium channel assay kit (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. As described earlier [31 (link),39 (link)], the 2X Fluo-4 Direct calcium reagent loading solution containing probenecid (5 mM) was added in equal volume to the 96-well plate containing 5 × 10⁴ HEK-293 (AddexBio, San Diego, CA) cells/well. The plates were allowed to equilibrate for 1 h at 37°C with 5% CO_2, and fluorescence (pre-stimulation) was immediately measured in a microplate reader. The cells were then treated with Ar1-Ar24 (16 μg/mL), Ca²⁺ channel inhibitor verapamil (32 μg/mL), and DMSO (mock) at 30 s. Then at 60 s, carbachol (50 μg/mL; calcium channel stimulator) was added, and the fluorescence (post-stimulation) was recorded for an additional 120 s.