The oxidative injury INS-1 cell model was established with H2O2 as described previously.30 (link) Briefly, INS-1 cells were inoculated with 1.5×104 cells/well in 96-well plates and were cultured for 72 h at 37°C with 5% CO2. H2O2 was added to the wells at a final concentration of 400 μM and the cells were incubated for 1 h. Then the cells were treated for 24 h in fresh media containing 10 nM of SCD (using the final concentration of DBAYL as the quantitative index). The cell proliferation rates were determined with MTT. Intracellular ROS levels were determined with fluorometric intracellular ROS kit (Sigma-Aldrich) using the method described previously.31 (link) For SCD (10 nM) treatment, the same doses of SeNPs, SC (210 nM, using the final concentration of SeNPs as the quantitative index), BAY55-9837, Ex-4, DBAYL (10 nM) or the same volume of PBS were used for controls.
For the receptor VPAC2 blocking experiments, cells were preincubated for 30 min at 37°C with 50 nM of a VPAC2-selective inhibitor PG99-465 prior to 10 nM SCD treatment.