Dnmt3a

Complete protease inhibitor Cocktail (Roche, Guangzhou, China) supplemented RIPA lysis buffer (Beyotime, Shanghai, China) was used to isolate sample proteins. Bicinchoninic acid protein assay kit (Peirce, Rockford, IL, USA) was employed to detect the concentration of total proteins. The extracted proteins were subjected to 10% SDS-PAGE, followed by electroblotting onto PVDF membranes (Millipore, Billerica, MA, USA). Blocked with 5% skimmed milk at 25°C for 2 h, the blots were incubated with the respective primary antibodies at 4°C overnight. Then, the blots were probed with corresponding HRP-conjugated secondary antibodies. Last, Enhanced Chemiluminescence Western Blotting kit (Peirce) was used to visualize the immunoreactive protein bands. The following primary antibodies were used in this section: DNMT3a (Bioss antibodies, Woburn, MA, USA), PCNA, caspase-3, cleaved caspase-3, MMP-2, and MMP-7 (Abcam, Cambridge, UK); PTEN, Akt, p-Akt, and β-actin (Cell Signaling Technology, Beverly, MA, USA). Each experiment was repeated independently at least 3 times.