Primary antibodies against β actin

Cytoplasmic proteins were isolated using a Cytoplasm Protein Extraction Kit. Thereafter, a BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, China) was used to determine the protein concentrations. The samples were boiled for 5 min. Next, portions containing 20 μg protein were separated on 12% SDS–polyacrylamide gel and transferred onto PVDF membranes (Millipore, Bedford, MA) at a current of 200 mA for 40 min. After blocking for 2 h in a TBS containing 0.1% Tween 20 (TBST) and 5% w/v skim milk powder at room temperature, these membranes were incubated overnight at 4 °C with primary antibodies against β-actin, cytochrome c, and Smac (1:300, BOSTER, Wuhan, China), and anti-p-JNK (1:2000, Cell Signaling Technology, Danvers, MA), with dilutions in TBST. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, BOSTER, Wuhan, China) for 1 h at room temperature and again washed three or four times. The bands were developed using an ECL kit following the manufacturer’s instructions, with β-actin serving as a loading control. The X-OMAT BT films (Carestream, Xiamen, China) were scanned and quantitated using Quantity One software.

Polyphyllin I (PPI) was purchased from Chengdu Mansite Pharmacetical CO, LTD (Purity ≥ 98%, lot number: MUST—16021905, Chengdu, China), and L-15 medium was purchased from Hyclone (SH30525.01, Logan, UT, USA). FBS was purchased from Gibco (Waltham, MA, USA). DMSO, acridine orange (AO) staining kit and mounting medium were purchased from Solarbio (Beijing, China). MTT and CQ were purchased from Sigma (St Louis, MI, USA). Primary antibodies against β-actin, LC3/Atg8, and mTOR were purchased from Boster (Wuhan, China). Primary antibodies against p-mTOR, AKT, p-AKT, and p53 were purchased from Cell Signaling. ROS assay kits were purchased from Beyotime (Chengdu, China).