P cdk1

Total proteins from cells were extracted using ice-cold lysis buffer (50 mM Tris-HCL pH 7.5, 150 mM NaCl, 1% NP40, 1 mM PMSF, and 10 units/ml aprotinin) for 5–10 min, then centrifuged at 12000 rpm form 10 min at 4 °C to obtain the whole cell lysate. Proteins (about 20 μg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes and incubated overnight at 4 °C with antibody against MCM4, MCM5, MCM6, and MCM7 (Santa Cruz), p-histone H3 (Thr3) (Cell Signaling technology), histone H3 (Cell Signaling technology), p-Cdk1 (Abcam) or GAPDH (Bioworld technology). After washing with Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with HRP-conjugated IgG at room temperature for 1 h. Signal detection was carried out with an ECL system (Millipore, Billerica, MA, USA). WB band were quantified with gel-pro software and expressed as optical density fold difference related to GAPDH (relative OD).

DMEM medium (Cat No. C11995) and FBS (Cat No.10099-141) were purchased from Gibco (USA). MTT reagent and propidium iodide (PI, Cat No. P8080) were obtained from Solarbio (China). The primary antibody of CDK1 (Cat No. ab133327), p-CDK1 (Cat No. ab201008), cyclin B1 (Cat No. ab32053), p21 (Cat No. ab109520), TGF-β (Cat No. ab215715), and Smad 2/Smad3 (Cat No. ab202445) and the secondary antibody were purchased from Abcam (USA). The dyes of DAPI (Cat No. 268298) were purchased from Sigma (USA). The ELISA assay kits of ROS (Cat No. E004-1-1), CAT (Cat No. A007-1-1), GSH (Cat No. A005-1-2), and SOD (Cat No. A001-3-2) were obtained from Nanjing Jiancheng Bioengineering Institute (China). The LY2157299 (Cat No. HY-13226), a TGF-β inhibitor, was purchased from MedChemExpress (MCE). The organic solvents ethanol, petroleum ether, chloroform, and ethyl acetate were purchased from Sinopharm Chemical Reagent Co., Ltd (China).