Pi rnase kit

For proliferation analysis, cells were treated with 10 μmol/L EdU (GeneCopoeia; CA, USA) for 4 h, harvested, and fixed with 4% (m/V) paraformaldehyde. EdU fluorescence signal was detected by flow cytometry. For cell cycle analysis, EdU-labeled cells were treated with PI/RNase kit (Beyotime Institute of Biotechnology; Haimen, China) for 30 min and subjected to flow cytometry.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from MP (Solon, OH, USA). Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, phosphate-buffered saline (PBS), fetal bovine serum (FBS), Pierce BCA protein assay kit, and Super Signal™ West Pico Chemiluminescent Substrate kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Calcein/propidium iodide (PI) kit, RIPA buffer, crystal violet, 4% paraformaldehyde, and PI/RNase kit were from Beyotime Biotechnology (Jiangsu, China). Trizol buffer was purchased from Invitrogen (Carlsbad, CA, USA). The primary antibodies against cyclin D1, c-Myc, PI3K, P-PI3K, Akt, P-Akt, HIF-1A, VEGFA, MAPK, and P-MAPK were purchased from Affinity (Cincinnati, OH, USA), and β-actin and GAPDH were from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies were obtained from Millipore Corporation (Temecula, CA, USA).