Western Blot

Cell and tissues lysates were collected as previously described (Nie et al., 2020). Protein concentrations were determined using BCA Assay (Beyotime Biotechnology). Equal amounts of protein were separated with 8–12% SDS-PAGE and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, United States). TBST containing with 5% nonfat milk or bovine serum albumin was used to block nonspecific binding for 2 h at room temperature. Then, membranes were incubated with primary antibodies according to the instructions overnight at 4°C followed by appropriate secondary antibodies. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (Tanon, Shanghai). Primary and secondary antibodies included: PPARγ (1:1,000, Santa cruze, #sc-7273), BCL-XL (1:1,000, Cell Signaling Technology, #2764), BAX (1:1,000, Cell Signaling Technology, #5023), BNIP3 (1:500, Cell Signaling Technology, #44060), LC3B (1:1,000, Abcam, #ab51520), P62 (1:1,000, Abcam, #ab109012), mTOR (1:1,000, Cell Signaling Technology, #2983), p-mTOR^Ser2448 (1:1,000, Cell Signaling Technology, #5536), ULK1 (1:1,000, Cell Signaling Technology, #6439), p-ULK1^Ser317 (1:1,000, Cell Signaling Technology, #12753), S6 (1:1,000, Cell Signaling Technology, #2217), P-S6^Ser235/236 (1:1,000, Cell Signaling Technology, #2211), ATG4D (1:400, Zen-bioscience, #507842), SOD2 (1:1,000, proteintech, #66474-1-Ig), GAPDH (1:5,000, Proteintech, #60004-1-Ig), CD44 (1:1,000, Cell Signaling Technology, #5640), CD133 (1:1,000, Sigma-Aldrich, #4300882).