Gc 1 cell line

The mouse-derived spermatogonia cell line (GC-1 cell line) was bought from American Type Culture Collection (ATCC) and maintained in DMEM medium (Gibco, NY, USA) supplemented with 10% FBS (Gibco, NY, USA). Primary antibodies, anti-Beclin1 and anti-LC3, were from Sigma-Aldrich; anti-GAPDH was from Bioss Biotechnology. Secondary antibodies for immunoblot analysis were from Bioss Biotechnology. ALC was from Santa Cruz Biotechnology. Cell counting kit-8 (CCK-8) solution was from Dojindo Molecular Technologies Institute. Catalase (CAT) (A007-1-1), malondialdehyde (MDA) (A003-1-2), and total antioxidant capacity (T-AOC) (A015-1-2) assay kits were from Nanjing Jiancheng Bioengineering Institute (http://www.njjcbio.com/product.asp?cid=27&sort=saleorder). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), monodansylcadaverine (MDC), 4′,6-diamidino-2-phenylindole (DAPI), and acridine orange/ethidium bromide (AO/EB) staining Kit were from Beyotime Biotechnology (https://www.beyotime.com/index.htm). Trizol reagent and Prime Script reverse transcriptase reagent kit were from TaKaRa Biotechnology.

GC-1 cell line from American Type Culture Collection (ATCC, Manassas, VA, USA) was maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 6% fetal bovine serum, FBS (Invitrogen). TCam-2 cell line (kindly provided by Prof. L. H. Looijenga, Erasmus MC-University Medical Center Rotterdam, Rotterdam, The Netherlands and P. Chieffi, University of Campania “Luigi Vanvitelli”, Caserta, Italy) was cultured in RPMI 1640 (Invitrogen) containing 10% fetal calf serum, FCS. Cell cultures were maintained in humidified 95% air and 5% CO₂ atmosphere at 37 °C. For treatments, 70% confluent cells were cultured in DMEM without phenol red and serum for one day and subsequently treated for 24 hours with 100 nM 17β-estradiol (E2), 10 nM 5α-dihydrotestosterone (DHT), 10 nM insulin-like growth factor (IGF-1) and 10 nM retinoic acid (RA) (Sigma-Aldrich Co., St. Louis, MO, USA).