P akt

The cultured SSCs were digested by RIPA (Beyotime, ShangHai, China) at 4 °C for 30 min and the protein were degenerated in 5×SDS sample loading buffer at 100°C for 10 min. Total protein was separated by SDS-PAGE 100V for 90 min, transferred to a 0.22-μm PVDF membrane at approximately 200 mA for 90 min, and incubated with B-ACTIN (1:1000, Beyotime, Shanghai, China), SOX9 (1:500, BOSTER, Wuhan, China), PCNA (1:1000, BOSTER), PLZF (1:300, Santa Cruz, USA), p-AKT (1:1000, Sangon Biotech, Beijing, China), AKT (1:1000, Sangon Biotech), ERK (1:1000, CST, Beverly, MA, USA), p-ERK (1:1000, CST). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse were used as a secondary antibody (1:2000, BOSTER). Detection was performed using the Thermo Scientific Pierce ECL western blot substrate (Thermo Scientific, USA). The results were analyzed with a Tanon-410 automatic gel imaging system (Tanon Corporation, Shanghai, China).

The reagents used in the study were procured as follows: RPMI-1640 and DMEM/F12 culture media (Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd.); premium fetal bovine serum (Sangon Biotech Co., Ltd.); CCK-8 assay kit (Vazyme Biotech Co., Ltd.); Transwell chambers (Corning Incorporated USA); antibodies for Akt (Phospho-Ser473 1:1000 Sangon Biotech Co., Ltd.), p-Akt (#4060, Ser 473, 1:1000 CST), mTOR (#7C10, 2983S 1:1000 CST), p-mTOR (#AF3308, Ser2448, 1:1000), p-PI3K (#AF3242, Tyr458, 1:1000), PIK3R1 (#A00318-1, 1:1000), and GAPDH (ab8245, 1:10000); AKT pathway activator SC79 (#CSN16878 Csnpharm).