Glc82

Normal lung cell line 2BS, lung cancer cell lines A549 and H1299 were gifts from Dr. Hongti Jia at the Department of Biochemistry and Molecular Biology, Peking University Health Science Center. A549 and H1299 cells were originally obtained from American Type Culture Collection (ATCC, Rockville, MD). 2BS cells were originally obtained from the National Institute of Biological Products (Beijing, China). Lung cancer cell lines GLC82 and Calu-3 for adenocarcinoma and NCI-H520 for squamous cell carcinoma were obtained from American Type Culture Collection (ATCC). The 2BS cells were cultured in DMEM (Invitrogen, Carlsbad, USA), and the other cell lines were propagated in RPMI 1640 medium (Invitrogen). The medium were supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. All cells were maintained in medium at 37°C in a humidified atmosphere of 5% CO₂.

The cell lines including SKOV3, OVCAR3, HeLa, SiHa, GLC-82, A549 and HepG2 were purchased from American Type Culture Collection (ATCC) and were cultured in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), streptomycin (100 mg/mL) and penicillin (100 U/mL) at 37°C in a 5% CO₂ humid incubator. DCA and Cycloheximide (CHX) were purchased from Sigma-Aldrich (Louis, MO, USA). Met, U0126, MG132 and Hoechst 33258 were purchased from Beyotime Company (Shanghai, China). MK2206 was purchased from Selleck Company (Shanghai, China). Caspase-3 Activity Assay Kit and Reactive Oxygen Species Assay Kit were purchased from Beyotime Company (Shanghai, China). Annexin V-FITC and PI were purchased from BD Bioscience (BD, NJ, USA). siRNAs to Mcl-1, ATG7, PDH and control siRNA were from GenePharma (Shanghai, China). pCMV and pcDNA3.1, pCMV-Mcl-1, pcDNA3.1-PDK1 and pcDNA3.1-PDK2 expression plasmids were bought from Obio Technology (Shanghai, China).

NSCLC cells (A549, H1299, LC-2/ad, GLC-82 and H520) and the normal lung cell line MRC-5 were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were then maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) containing 10% fetal bovine serum (Gibco) at 37°C in a humidified incubator with 5% CO₂.

uPAR inhibitors C6 and C37^{22 (link)} were obtained from the NCI/DTP Open Chemical Repository, were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C, at a concentration of 0.01 mol/L. For this study, a panel of immortalized NSCLC (PC9, HCC827, GLC82, H460, H1299, A549) and CRC (SW48, LS174T, HCT116, GEO, SW480, LoVo) cancer cell lines, obtained from the American Type Culture Collection, were used. Some experiments were performed on SW48 cells stably transfected with different pRcCMV plasmids containing KAS wild-type or mutant coding sequences. For a detailed description, see “RAS gene mutagenesis and RAS mutant transfection” methods and supplementary methods.