Glucose stimulated insulin secretion (GSIS) and calcium influx assays

For GSIS assays, MIN6 cells were initially seeded into 96 well plates in high glucose DMEM media 12 h before experiments. Rab1A siRNA and controls were transfected using lipofectamine 3000. In the meantime, Actinomycin D (Sigma Aldrich, A9415) or DMSO as control were added into at 1 μg/mL. 12 h later, the cells were proceeded for GSIS assay as described elsewhere. Briefly, cells were pre-incubated with DMEM media (no FBS, no glucose) for 2 h. Then the cells were incubated with DMEM media containing 1mM glucose. After incubation for 30 min, the cells were incubated with DMEM media containing 3 mM or 20 mM glucose for 1 h (basal: 3 mM; stimulatory: 20 mM). The supernatant from different wells was collected for further ELISA analysis (Mercodia Rat/Mouse ELISA, Sweden).

For calcium influx measurements, experiments were performed in size-matched isolated pancreatic islets or control/Rab1A knockdown MIN6 cells. Briefly, 8 islets of 150–200 μm diameter or MIN6 cells kept in 96-well plates were pre-incubated for 1 h in Krebs-Ringer bicarbonate (KRB) buffer containing (in mM): 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, 25 mM NaHCO₃ supplemented with 0.5% bovine serum albumin and 1 mM glucose. Islets or Cells were loaded with the Ca²⁺-sensitive dye Fluo-4 followed manufacturer’s instructions (Fluo-4 Direct Calcium Assay Kit, {"type":"entrez-nucleotide","attrs":{"text":"F10471","term_id":"683129","term_text":"F10471"}}F10471, Thermo Fisher). After incubation for calcium dye loading, the calcium signal from 1 mM glucose KRB buffer was measured as the basal value. Then, the islets or cells were transferred to KRB buffer with 20 mM glucose and 0.5% BSA. The calcium influx was measured using Tecan Infinite M200 microreader (Tecan Trading, model: Infinite M200) as previously described (Martínez et al., 2017). The data was calculated using the average of first 9 cycles (unstimulated) as baseline.