P30 330250

HEK293T, TM4, GC-1, and HeLa cells obtained from China Center for Type Culture Collection. The cells were cultured in high glucose DMEM (SH30022.01B, HyClone, Logan, USA) with 12% FBS (P30-330250, PAN-Biotech, Aidenbach, Germany) at 37 °C in a 5% CO₂ in cell incubator. For transfection, cells were cultured in cell plates or glass cover slides. Lipofectamine 2000 (11668027, Invitrogen) was used in each well. For starvation treatments, the cells were cultured in EBSS (Cat# SH30029.02, HyClone, Logan, USA) for various times.

Gonads were collected and washed twice in 1xPBS. Sex was verified by histological analysis of the gonad sections. The sections were stained by hematoxylin and eosin and images were captured using a Leica microsystem (Leica). Three samples were minced with sterilized scissors after washing three times with D-Hank’s, then were digested in collagenase type IV (C5138, Sigma-Aldrich, St Louis, USA) of 1 mg/ml at 28 °C for 20 min, with shaking up and down once in every 3 min. The tubes were spun at 300g and the supernatant was removed. The samples were washed three times with D-Hank’s, then digested with 0.25% trypsin (1541894, gibco, Grand Island, NY, USA) and Deoxyribonuclease I (EN0521, Thermo Scientific, Rockford, IL, USA) buffered by Leiboviz’s L-15 (SH30525.01, HyClone, Utah, USA) medium for 10 min, with shaking up and down once in every 2 min. The digestion was stopped with Leiboviz’s L-15 medium supplemented with 10% FBS (P30-330250, PAN-Biotech, Aidenbach, Germany). After filtering by strainers with mesh sizes of 70 μm (15-1070, Biologix, Jinan, Shandong Province, China) and 40 μm (15-1040, Biologix), single cells were obtained. The cells were pelleted by centrifugation at 300g for 5 min and resuspended with Leiboviz’s L-15 medium supplemented with 10% FBS. The cell viability was evaluated by trypan blue (T6146, Sigma–Aldrich) staining with a hemocytometer.

HEK293T and CHO cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (SH30022.01B, HyClone, Logan, USA) with 10% fetal bovine serum (FBS) (P30-330250, PAN-Biotech, Aidenbach, Germany) in 12/48-well plates and Lipofectamine^TM 2000 (11668027, Invitrogen) was used for transfection according to the routine protocol. For luciferase assays, cells per well was transfected with 0.4 μg recombinant constructs and 10 ng pRL-TK (E2241, Promega). Then luciferase activities were measured by a dual-luciferase reporter assay system (Promega, Madison, WI, USA) and a Modulus Single Tube Multimode Reader (Turner Biosystems, Sunnyvale, CA, USA) according to the manufacturer’s protocol. The experiments were repeated at least 3 times, and the results were expressed as the means ± SD.