Slc3a2

Western blotting and the analysis of the blot results was conducted as previously reported^{27 (link)}. In brief, cells were lysed and centrifuged at 12,000 g for 10 minutes at 4 °C. The supernatants were denatured at 100 °C for 5 minutes and mixed with a 1/5 volume (corresponding to the volume of the supernatants) of loading buffer (Beyotime, China). Then, 30-µg protein samples were loaded into each well in a 10% SDS-polyacrylamide gel and were subjected to electrophoresis. Once the proteins of diverse molecular weights were sufficiently separated, the proteins were transferred to PVDF membranes (Millipore, USA). Next, the PVDF membranes with the transferred proteins were blocked with 5% nonfat milk diluted in TBST for 2 hours at 25 °C and were then incubated with primary antibodies, such as SLC7A11, SLC3A2, GPX4, GSS, GCLC, ACSL4 (Cell Signaling Technology, USA) and β-actin (Beyotime, Shanghai, China), with dilutions of 1:1500, 1:2000, 1:2000, 1:2500, 1:3000 and 1:10,000, respectively, at 4 °C for 12 hours. Next, the membrane was washed again with TBST and was incubated with a 1:10,000 dilution of horseradish peroxidase (HRP)-labelled anti-rabbit IgG (Beyotime, Shanghai, China) for 1 hour at 25 °C. Finally, the respective protein bands were visualized using enhanced chemiluminescence reagents (BOSTER, Wuhan, China).

LPS (E. coli 0111:B4), dimethyl sulfoxide (DMSO), Tween-80, Hoechst 33342 and amino acid transporter inhibitor BCH (2-amino-2-norbornanecarboxylic acid) were bought from Sigma-Aldrich (St. Louis, MO, USA). Thioglycollate medium (Brewer modified) was obtained from Becton Dickinson (Sparks, MD, USA). Piperine was purchased from Guangzhou Institute for Drug Control (Guangzhou, China), dissolved in DMSO and stored at −20°C. Rabbit antibodies against phospho(p)-p70S6K, p70S6K, p-4E-BP1, 4E-BP1, p-S6(Ser235/236), GATA6, cleaved caspase-3, SLC7A5, SLC3A2, mTOR, and β-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). The mouse antibody against LAMP2 was obtained from Abcam (Cambridge, MA, USA). PE-F4/80, FITC-CD11b, AlexaFluor488-CD11b, and APC-MHCII were obtained from eBioscience (San Diego, CA, USA). DMEM medium, fetal bovine serum (FBS), penicillin and streptomycin were products of Invitrogen (Carlsbad, CA, USA).
Female C57BL/6 mice were bought from the Experimental Animal Center of Southern Medical University (Guangzhou, China). Animal experiments were designed following National Institutes of Health guidelines and were approved by the Committee on the Ethics of Animal Experiments of Jinan University.