Cell culture, flow cytometry and immunoblotting
Rat insulinoma INS-1 cells were cultured in RPMI-1640 medium (Invitrogen), supplemented with 10% heat inactivated fetal bovine serum (HyClone/Thermo Fisher Scientific), 1 mM sodium pyruvate, 2 mM L-glutamate, 0.05 mM 2-mercaptoethanol, 10 mM HEPES and penicillin/streptomycin. Cells were transfected with Smart Pool Cdk2 siRNA or Cdk4 siRNA or control dsRNA (Dharmacon/Thermo Fisher Scientific), using the RNAiMax lipofection reagent (GiBCO/Invitrogen). For stimulation of cell cycle progression, cells at 48 h posttransfection were incubated in the medium with low (0.1 mM) glucose for 48 h, followed by incubation in the medium with high (11 mM) glucose. At various times after stimulation with high glucose, cells were harvested for immunoblotting and flow cytometry. At 30 min before each harvesting point, bromodeoxyuridine (BrdU, Sigma/Aldrich) was added to the medium. Immunoblotting and flow cytometry were performed as described previously 14. Anti-BrdU antibody for flow cytometry was obtained from Pharmingen/BD Biosciences. Antibodies against CDK2, CDK4, CDK6, Cyclin D1, Cyclin D3, Cyclin E and Actin were purchased from Santa Cruz Biotechnology. Anti-total RB antibody was from Pharmingen/BD Biosciences, and the phospho-specific antibodies against RB and p130 were from Cell Signaling Technology.