RSC96 Culture and Treatment

The RSC96 SC line was obtained from ScienCell Research Laboratories. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin solution (P/S) in a humidified incubator (37°C, 5% CO₂). After two passages, the cells were seeded in 96-well plates (5 × 10³ cells/well), and various concentrations of FGF10 (0.043, 0.43, 4.3, 43 nm) were added for 2 h. Then, the medium was supplemented with 100-μm H₂O₂ for another 2 h. To further evaluate the effect of PI3K/Akt activation on oxidative injury, cells were pretreated with the PI3K inhibitor LY294002 (20 μm) (Wang et al., 2012) for 2 h before the addition of FGF10.

To evaluate cell survival, a cell counting kit (CCK-8, Beyotime Institute of Biotechnology, China) was used. The test was performed according to the manufacturer’s instructions. In brief, 10 μl of CCK-8 solution were added to each well, and the cells were incubated for 2 h at 37°C. The optical density was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA). All experiments were repeated at least three times.