Fibroblast growth factor 2 (fgf2)

Primary MEF cells were isolated from embryos at E13.5 stages and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum as described (28 (link)). The cells were starved for 24 to 48 hours before FGF2 (104-02-50, ScienceCell) or IGF1 (291-G1-200, R&D Systems) treatment at 37°C. For gene deletions, MEF cells carrying homozygous flox alleles were infected with Ad5CMV-eGFP or Ad5CMVCre-eGFP (Gene Transfer Vector Core, University of Iowa, IA) in DMEM overnight at a multiplicity of infection of 500 plaque forming units per cell and cultured for 5 days. For inhibitor studies, cells were starved for 24 hours and treated for another 6 hours with PI3K inhibitors LY294002 (50 μM; #9901) and PX866 (1 μM; #13055), MEK inhibitor U0126 (50 μM; #9903), or mTOR inhibitor Torin (150 nM; #14385), all from Cell Signaling Technology. After growth factor stimulation, cells were washed in cold phosphate-buffered saline and harvested in ice-cold CelLytic reagent (C2978, Sigma-Aldrich) supplemented with protease inhibitor cocktail (78841, Thermo Fisher Scientific). Protein samples were denatured at 95°C for 5 min before loaded onto SDS–polyacrylamide gel electrophoresis gels. The antibodies used for Western blot were the same for immunohistochemistry, except anti-pERK1/2 (sc-7383, from Santa Cruz Biotechnology) and anti-ERK1/2 (#4695, Cell Signaling Technology).

HTM cells (ScienCell Research Laboratories, Inc.) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 15% fetal bovine serum, 2 mM L-glutamine, 0.05% gentamicin, 1 ng/ml FGF-2 and 0.25 µg/ml amphotericin B (all from ScienCell Research Laboratories, Inc.) at 37°C with 5% CO₂. The medium was replaced every day when the culture reached 70% confluence. To induce oxidative stress, cells were incubated in DMEM supplemented with H₂O₂ (Beyotime Institute of Biotechnology) at concentrations of 100, 200, 300, 400 µM for 24 h after reaching 85% confluence.