F12k culture medium

The study used the following reagents: paraquat (Sigma-Aldrich, United States); metformin (Sigma-Aldrich, United States); hematoxylin and eosin (HE) staining reagent (Department of Pathology of Zhengzhou University, China); Rat interleukin (IL)-6 (No.70-EK306/3-96), tumor necrosis factor (TNF)-α (No.70-EK382HS-96), and IL-10 (No.70-EK310/2-96); enzyme-linked immunosorbent assay (ELISA) kits (Multi Sciences (LIANKE) Biotechnology Company, China); fetal bovine serum (Gibco, United States); F12K culture medium (Sigma-Aldrich, United States); cell counting kit (CCK-8) assays (Dojindo, Japan); bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, United States); cell lysis solution (GBCBIO, Guangzhou, China); quantitative polymerase chain reaction (q-PCR) kit (TaKaRa, Dalian, China); polyvinylidene fluoride membrane (Millipore, Bedford, MA); inducible nitric oxide synthase (iNOS) (SC-7271) and arginase 1 (Arg1) (SC-271430) (Santa Cruz Biotechnology, United States); and β-actin (13E5) (Cell Signaling Technology, United States).

The NSCLC cell lines A549 and H1299 were all obtained from Chinese Academy of Sciences Shanghai Branch Cell Bank (Shanghai, China). A549 cells were cultured in F12K culture medium (Sigma–Aldrich, St. Louis, MO, U.S.A.) supplemented with 2.5 g/l NaHCO₃ and 10% (v/v) fetal bovine serum (FBS; Sigma–Aldrich), while H1299 cells were seeded in Roswell Park Memorial Institute (RPMI) 1640 culture medium (Sigma–Aldrich) supplemented with 1.5 g/l NaHCO₃, 2.5 g/l glucose, 0.11 g/l sodium pyruvate and 10 % (v/v) FBS at 37°C in 5% CO₂ atmosphere. Until the cells grew into logarithmic phase, they were washed three times by PBS, digested with trypsin and then repeatedly beaten to single cell suspension. Subsequently, cells were seeded into a six-well plate in preparation for the following experiments.
Nicotine was purchased from Sigma–Aldrich (product number N3876) and added to cells after dilution with a concentration of 0, 1, 10, 100 and 200 μg/ml. After 48 h, LINC00460 expression level in A549 and H1299 cells was detected using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Time-dependent analysis was also measured as above instructions using 100 μg/ml.