3 methyladenine 3 ma

Peptide FLPNF (Figure 1), D-ANFLVH and NFGAIL, were synthesized by ChinaPeptides Co., Ltd (Shanghai, China, purity >98%) as previously described (Shi et al., 2019 (link)). Cell-penetrating peptide HIV-TAT(48–57) (GRKKRRQRRR) was fused to peptide FLPNF at the N terminal to enhance cellular permeability, and the TAT linked FLPNF was more effective in inducing autophagy (Supplementary Figure 1). The linker GSG (Gly–Ser–Gly) was added between TAT peptide and peptide FLPNF (GRKKRRQRRR-GSG-FLPNF) to prevent potential interaction between the two peptides and enhance peptide flexibility (Sutton et al., 2019 (link)). The synthetic peptides were solubilized in dimethyl sulfoxide (DMSO) at 100 mM according to the manufacturer’s instructions. Rapamycin, 3-Methyladenine (3-MA), and bafilomycin-A1 (Baf-A1) were obtained from Solarbio (Beijing, China). Insulin-like growth factor-1 (IGF-1) and protein phosphatase 2A (PP2A) inhibitor okadaic acid (OA) were purchased from sigma (85580C and O8010, respectively). Anti-β-actin, anti-Bax, anti-phospho-AKT, and anti-AKT antibodies were purchased from Proteintech, while anti-Bcl-2 antibody was from Santa Cruz Biotechnology. All other primary antibodies were obtained from Cell Signaling Technology (CST) if not indicated otherwise.

TPPU (Sigma‑Aldrich, Merck KGaA, Darmstadt, Germany) was dissolved in dimethyl sulfoxide (DMSO, Coolaber, Beijing, China) as a stock solution (10 mg/ml), and further diluted to 10 μM TPPU for cell treatment in vitro or 20 μM TPPU for local injection in vivo^{26 (link),47 (link)}. Cells were treated with or without an autophagy inhibitor 3-methyladenine (3-MA, 10 mM; Solarbio, Beijing, China), an autophagy inducer Rapamycin (Rapa, 5 µM; Solarbio, Beijing, China).