Mhy1485

A large yellow croaker hepatocyte line was obtained from the Fisheries College of JiMei University (Xiamen, Fujian, China). The hepatocytes were cultured in six-well plates at 1.5 × 10⁶ cells per well with Dulbecco’s modified Eagle’s medium (DMEM)/F12. After 2 d of culture, the hepatocytes were serum-starved with FBS-free DMEM/F12 for 2 h prior to receiving the experimental treatments. To elucidate the possible mechanism by which LA activates autophagy and antioxidation in hepatocytes, corresponding activators and inhibitors of the signaling pathways were used, including AICAR (an AMPK activator, S1802; Selleck Chemicals, Houston, TX, USA), CC (an AMPK inhibitor, S7306; Selleck Chemicals), MHY1485 (a TOR activator, B5853; APExBIO Technology, Houston, TX, USA) and RAPA (a TOR inhibitor, V900930; Sigma-Aldrich, St.Louis, MO, USA). To determine the mechanisms of autophagy and antioxidation turnover, hepatocytes were exposed to the pharmacological autophagy–lysosome pathway activator RAPA, the autophagy–lysosome pathway inhibitor 3-MA (HY-19312; Monmouth Junction, NJ, MCE) or CQ (HY-17589; MCE), the Nrf2 pathway activator NK-252 (HY-19734; MCE), or the Nrf2 pathway inhibitor ML385 (HY-100523; MCE).

The ovary tissue of patients (n = 11) of non-ovarian factors containing primordial follicles were collected using excision surgery at the Department of Obstetrics and Gynecology in Shanghai Changzheng Hospital. All the participants are transsexual patients that had normal ovarian function and were not under hormonal treatment before the surgery. This study is approved by the Institutional Medical and Ethical Review Committee of Second Military Medical University. Clinical characteristics of cases were shown in Table 1. After being cut into 1 mm³ cubes, cortical tissues were placed on culture plate inserts (Millipore) and cultured in 400 μl of DMEM/F12 (Gibco) containing 10% human serum albumin (CSL Behring), 1% penicillin-streptomycin solution (HyClone), 0.3 IU/ml FSH, and 0.05 mg/ml L-ascorbic acid under a membrane insert to cover ovaries with a thin layer of medium. Ovaries were treated with 1–20 μM MHY1485 (10 mM in 1 ml DMSO, APExBIO, United States) and cultured for 3 h before immunoblotting analysis. Control ovaries were treated with solvent only. Other pairs of ovaries were cultured for 2 days with a medium change after 24 h of culture. Ovaries were fixed with formalin for histological analysis or prepared for transplantation before weighing.

T-2 toxin was purchased from BBI (Markham, ON, Canada). [4,5-Dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Munich, Bavaria, Germany). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT, USA). Dulbecco’s modified eagle medium (DMEM)/nutrient mixture (Ham’s) F-12 with HEPES and horse serum was purchased from Gibco (Carlsbad, CA USA). The Annexin V-FITC apoptosis detection kit was purchased from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China). The primary antibodies for p-mTOR ser473 and p-mTORser2448 were obtained from Abcam (Cambridge, Cambridge, UK). The primary antibodies of mTOR, p-AKTser473, AKT, caspase-3, and β-actin were obtained from Proteintech (Chicago, IL, USA), and the signal was visualized by using HRP-conjugated secondary antibodies that were obtained from Proteintech. BAPTA-AM and MHY1485 were purchased from ApexBio (Houston, TX, USA).