3 methyladenine 3 ma

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin was purchased from Qilu Pharm (Jinan, China). 3-Methyladenine (3-MA) and N-acetylcysteine (NAC) were obtained from MedChem Express (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) high glucose were purchased from Gibco (Carlsbad, CA, USA). The antibodies used included Bax (5023), Bcl-2 (4223), cleaved caspase-3 (9664), Beclin-1 (3495), LC3B (3868), SQSTM1/p62 (39749), SAPK/JNK (9252), phospho-SAPK/JNK (Thr 183/Try 185, 4668), phospho-AKT (Ser 473, 4060), and phospho-mTOR (Ser 2448, 5536), all of which were acquired from Cell Signaling Technology (Danvers, MA, USA). Mouse anti-human GAPDH, rabbit anti-human β-actin, and secondary antibodies including goat anti-rabbit and goat anti-mouse were purchased from Proteintech (Wuhan, China).

Eicosapentaenoic Acid (purity, ≥97%), 3-Methyladenine (3-MA, a PI3K inhibitor) and Dorsomorphin (Compound C, an AMPK inhibitor) were purchased from MedChemExpress (NJ, United States). Type II collagenase, TBHP, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, United States). Primary antibodies for cleaved-caspase 3 (C-C3), Beclin-1, LC3B, p-mTORC1, mTORC1, BCL-2, AMPK and p-AMPK were procured from Cell Signaling Technologies (Danvers, MA, United States). Antibodies against BAX, BCL-2, Collagen II, MMP13 and P62 were purchased from Abcam (Cambridge, United Kindom). Antibodies against p-PERK, PERK, p-eIF2α, eIF2α, CHOP, ADAMTS5, Aggrecan, and ATG5 were purchased from ABclonal (WH, CHINA). Antibodies against ATF4, GRP78, Collagen II, and β-actin were purchased from Proteintech (NJ, China). Horseradish peroxidase-labeled secondary antibodies, Alexa Fluor^® 488-labeled goat anti-rabbit IgG (H + L) secondary antibody, and Alexa Fluor^® 594-labeled goat anti-mouse IgG (H + L) secondary antibody were purchased from Abcam. Further, 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (SH, CHINA). The reagents for cell culture were obtained from Gibco (Grand Island, NY, United States).

Cepharanthine was bought from Aladdin Chemistry Co., Ltd (C102706-5q), solubilized in DMSO, and stored at −20°C for later use in experiments. DMEM/F12 medium, FBS, pancreatic enzyme, and streptomycin/penicillin were acquired from Gibco (NY, United States). Collagenase II and DMSO were bought from Sigma-Aldrich (Merck KGaA, MO, United States). 17β-oestradiol (E2) was obtained from Selleck (Shanghai, China). Recombinant IL-1β and TNF-α were obtained from R&D Systems (Abingdon, United Kingdom). The radio-immuno precipitation assay (RIPA) lysis buffer and the bicinchoninic acid (BCA) assay kit were purchased from Hangzhou Fude Biological Technology Co., Ltd (Hangzhou, China). Also, 3-methyladenine (3-MA) and anisomycin were obtained from MedChemExpress (Monmouth Junction, NJ).

Salvia miltiorrhiza Bunge and Radix Puerariae were purchased from Changda Prepared Chinese Medicinal Herbs Co. Ltd (Anguo, China). Healthy SD rats, SPF clean grade, female, 10 weeks old, weight 200–250 g, were purchased from Chinese Academy of Sciences; The ELISA kits for detecting blood urea nitrogen (BUN), creatinine, ALP, OPG and RANKL were purchased from Wuhan Huamei Biological co., LTD (Wuhan, China); DCFH2-DA, Fluo-3/AM, BAPTA-AM and Hoechst 33,258 were obtained from Molecular Probes (Eugene, OR); Chemicals used for DPI, TTRA, AA, ALL, NAC, NDGA, Rot were purchased from Sigma-Aldrich (St. Louis, MO). H&E and Masson dying kits were purchased from Nanjing Jiancheng Biological Engineering Research Institute (Nanjing, China); 3-methyladenine (3-MA), and monodansyl cadaverine (MDC) were obtained from MedChemExpress; TRAP staining kit was obtained from BIO-SCIENCE COMPANY LIMITED (Shanghai, China); Antibodies GAPDH, LC3B, Beclin-1, p62, p-p65 and NF-κBp65 were purchased from Sigma Company; Antibodies NFATc1 and c-Fos were purchased from Chengdu Zen Bio (Chengdu, China); Secondary antibodies were purchased from American CST Company; Hoechst 33,342 was purchased from Thermo Fisher Scientific; JYB1-1 calcium removal solution and protein extraction kit were purchased from Solarbio Company (Beijing, China); RANKL was purchased from Sino Biological Company (Beijing, China).

Human 143B and U2OS osteosarcoma cells were purchased from Procell Life Science and Technology (Wuhan, China). Osteosarcoma cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and were incubated with 5% CO₂ at 37 °C. For lentivirus transfection, we constructed RILP overexpression (LV-RILP), RILP knockdown (shRILP), and empty control (LV-Control and shNC) using a lentivirus obtained from OBiO (Shanghai, China). 5 µg/mL puromycin were used to screen out stably constructed osteosarcoma cells (Yang et al. 2023 (link)). In addition, 740Y-P (30 µg/mL) (Wang et al. 2020a (link), b (link)) and 3-methyladenine (3-MA) (5 mM) (Lei et al. 2021 (link)) were purchased from MedChemExpress (USA).

The following primary antibodies were used in this study: anti-PIWIL1 antibody (ab12337; Abcam, Cambridge, UK), anti-p-mTOR antibody (ab109268; Abcam), anti-LC3 I/II (#2775S; Cell Signaling Technology, Danvers, MA, USA), anti-P62/SQSTM1 (#8025S; Cell Signaling Technology), anti-Parkin (#4211; Cell Signaling Technology), anti-phosphor-NAK/TBK (ab109272; Abcam), anti-NAK/TBK (A5497; Bimake), anti-optineurin (ab213556; Abcam), anti-β-actin (#4970; Cell Signaling Technology), anti-Akt (#4691; Cell Signaling Technology), anti-phospho-Akt (Ser473) (#9271; Cell Signaling Technology), anti-Ki67 (#4685; Cell Signaling Technology), anti-Bcl-2 (A5010; Bimake), anti-OCT4 (60242-1-Ig; Proteintech), and anti-Nanog (#4903T; Cell Signaling Technology). Goat anti-mouse and goat anti-rabbit antibodies (Biosciences) were used as the secondary antibodies for western blotting. Dexamethasone and doxorubicin were purchased from Sigma (St. Louis, MO, USA), and bortezomib was purchased from Selleck Chemicals. 3-Methyladenine (3-MA) and the mitophagy inhibitor cyclosporin A (CsA) were purchased from MedChemExpress (NJ, USA). All were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C for up to 6 months. For all cell-based experiments, drugs were diluted at least by 1:1000 to ensure that the final DMSO concentration was lower than 0.1%.