Bafilomycin a1

Elaiophylin was prepared from the deep-sea-derived Streptomyces sp. SCSIO 1934 [44 (link)]. Rapamycin, torin1, bafilomycin A1, compound C, STO-609, and rosiglitazone were purchased from Selleck. Insulin, IBMX, and dexamethasone were purchased from Sigma-Aldrich. Antibodies recognizing phospho-AMPKα (#2535), AMPKα (# 2532), phospho-ACC (#3661), ACC (#3662), phospho-AKT (#4060), phospho-S6K (#9204), S6K (#9202), 4EBP1 (#9644), phospho-ERK (#4370), phospho-c-JUN (#3270), and LC3B (#3868) were procured from Cell Signaling. Other antibodies included anti-AKT (Proteintech, 60203-2-Ig), anti-GST (Proteintech, 10000-0-AP), anti-TXNIP (Abcam, ab188865), and anti-ACTIN (Genescript, A00702).

DMSO was used as vehicle control. The proteasome inhibitor MG132 was obtained from Merck/Millipore and used at the final concentration of 20 μM for 3 h. PHD inhibitors IOX2 and FG-4592 were purchased from Selleckchem. VHL inhibitors VH032, VH298, and nonbinding epimer cisVH298 were synthesized by a group member of our laboratory (13 (link), 14 (link)). Compounds were added to cells for indicated length of time. Chloroquine (Merck) and bafilomycin A1 (Selleckchem) were added to cells for 3 h at 50 μM and 50 nM, respectively.

Unless noted, all compounds were prepared as stocks in DMSO (Synth, Diadema, SP, Brazil). Seriniquinone (SQ1) and its synthetic analogues SQ2, SQ3, SQ4, and SQ5 (Figure 1A) were obtained as described in [2 (link)] and were used with ≥98% purity. All other compounds were obtained from commercial sources, as follows: doxorubicin (DOX; Sigma-Aldrich, St. Louis, MO, USA), rapamycin (RAPA; Sigma-Aldrich, St. Louis, MO, USA), bafilomycin A1 (Selleck Chemicals, Houston, TX, USA), and Z-VAD-FMK (Cayman Chemical Company, Ann Arbor, MI, USA).

Suffruticosol C and resveratrol were isolated from the seeds of the tree peony. The concentration was chosen based on previous studies [27 (link),28 (link),31 (link),32 (link)] and considering that suffruticosol C is a trimer of resveratrol. The secondary antibodies and Erastin (E7781) were provided by Sigma-Aldrich (St. Louis, MO, USA). RSL3 (1219810-16-8) was obtained from Cayman (Ann Arbor, MI, USA), and Bafilomycin A1 (S1413) was obtained from Selleck Chemicals (Houston, TX, USA). PBS and trypsin were obtained from HyClone (Logan, UT, USA). The RPMI 1640 and DMEM media, streptomycin, β-mercaptoethanol, penicillin, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). The TB Green qRT-PCR kit (RR820A), PrimeScript™ RT reagent Kit (RR047A) and TRIzol were provided by Takara (Dalian, China). The primary antibodies used are listed as follows: Bcl-2 (12789-1-AP), Bax (50599-2-Ig), LC3II (18725-1-AP), p62 (18420-1-AP), and Actin (20536-1-AP) were purchased from Proteintech (Rosemont, IL, USA); S6 (2217S), p-S6 (4858S), AKT (9272), pT389-S6K (9234S/L), S6K (9202S), and Cleaved Caspase-3 (D175) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Ovaries were separated by microdissection from the mesonephros or ovarian capsule in prechilled PBS (10 mM, pH 7.4) under a stereomicroscope (ZSA302, COIC). The isolated ovaries were cultured in 6‐well culture dishes (NEST Biotechnology) with basic DMEM/F‐12 medium (Gibco, Life Technologies) and Penicillin‐Streptomycin at 37°C in a 5% CO₂, 95% air atmosphere with saturated humidity.
Ovaries were cultured in either medium with dimethylsulfoxide (DMSO) or medium supplemented with inhibitor. The concentration of inhibitors used in this study: GSK‐LSD1‐2Hcl (S7574; Selleck): 64 μM; 3MA (S2767; Selleck, USA): 2.5 mM; Z‐VAD‐fmk (S7023; Selleck): 50 μM; Chloroquine diphosphate (C6688; Sigma): 10 μM; Bafilomycin A1 (S1413; Selleck): 60 nM; MG132 (S2619; Selleck): 5 μM; Cycloheximide (HY‐12320; MedChemExpress): 2 nM; and SP2509 (S7680; Selleck): 5 nM.