Galectin 3

Trypsin and bafilomycin A1 were purchased from Sigma Chemical Co., (St. Louis, MO, USA). Anti-GAPDH monoclonal antibody from Calbiochem-Merck Chemicals Ltd. (Nottingham, UK). MnSOD, catalase and OGG-1 antibodies from Adipogen (San Diego, CA, USA). Phospho-mTOR, mTOR, phosphor-AKT, AKT, LC3, p62, LAMP-I, Cathepsin B, HexA and galectin-3 were obtained from Cell Signaling Technology. A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN, USA). Grace’s insect medium was purchased from Gibco. The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA, USA).

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).

Cell homogenates and SDS-polyacrylamide gels for electrophoresis were prepared as described previously [8 (link)]. The membranes were incubated with different primary antibodies [GRP78 (1:1000) (Abcam, UK), caspase-3 (1:1000) (Cell Signaling Technology, USA), Wnt1 (1:1000) (Abcam, UK), galectin-3 (1:1000) (Cell Signaling Technology, USA), BMP-2 (1:1000) (Abcam, UK), and β-actin (1:2000) (Anhui, China)] overnight at 4 °C and visualized with the corresponding secondary antibody at room temperature for 1 h. Protein bands were visualized using enhanced chemiluminescence (ECL) (Millipore, Billerica, MA, USA), and the results were quantified using Image-Pro Plus 6.0 software and normalized to β-actin. The experiment was repeated three times.