Murine cytokines

Gefitinib, simvastatin, and fluorescent dyes were obtained from Melone Pharmaceutical (Dalian, China). Osimertinib was from Selleck Chemicals (Texas, USA), and murine cytokines were from Peprotech (New Jersey, USA), and LPS from Sigma-Aldrich (St. Louis, USA). Soybean phosphatidylcholine (SPC), cholesterol, and DSPE-PEG₂₀₀₀, DSPE-PEG-NHS, and DSPE-PEG-Mal were obtained from Advanced Vehicle Technology (Shanghai, China). The derivative T12 peptide (sequence: CGGGTHRPPMWSPVWP) was synthesized by Bankpeptide Biological Technology (Hefei, China). The primary antibodies of EGFR, phospho-EGFR (Tyr1068), Erk1/2, phosphor-Erk1/2 (Thr202/Tyr204), Akt, phosphor-Akt (Ser473), VEGFR2, VEGF, caspase3, cleaved-caspase3, LC3, TGF-β, GAPDH, and galectin-3 were purchased from Cell Signal Technology (Boston, USA). The primary antibody β-Actin was obtained from Sigma-Aldrich (St. Louis, USA). The primary antibodies of CD206 and TNF-α were from Abcam (Cambridge, UK). Anti-HIF-1α was obtained from Novus (Shanghai, China) and anti-IFN-γ was from Absin (Shanghai, China). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse lgG secondary antibody was obtained from Beyotime (Shanghai, China). All other reagents (analytical grade) were provided by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).

The mAITL tumor cells or WT splenocytes were cultured at 5E5 cells/well in RPMI media supplemented with 10% FCS, 50 nM β-Mercaptoethanol and following murine cytokines obtained from Peprotech: 25 ng mL⁻¹ IL-6, 50 ng mL⁻¹ IL-21, 10 ng mL⁻¹ IL-7, 10 ng mL⁻¹ IL-15 and 5 ng mL⁻¹ IL-2. MN58b (5 μM) was added to the medium for 72 h as indicated. Etomoxir was added at the indicated doses to the cells in the medium described above in the absence or presence of N-Acetyl-L-cysteine (NAC, 2 mM) for 96 h. Ranolazine was added at the indicated doses to the tumor cells and medium described above for 96 h. DAPI staining was performed to evaluate cell death by FACS after surface-staining for CD4/CD8/PD1/CD19 using antibodies described above.