Dulbecco s modified eagle s medium dmem

The HepG2, Hep3B, H22, and 293T cell lines were given as gifts by Professor Aiguo Wang (Dalian Medical University, Dalian, China), who purchased them from the Chinese Academy of Sciences, Beijing, China. The cells were grown in McCoy’s medium (Sangon Biotech, Shanghai, China), RPMI-1640, or Dulbecco’s modified Eagle’s medium (DMEM) (Sangon Biotech, Shanghai, China) with 10% fetal bovine serum (FBS) (CellMax, AusGeneX, Queensland, Australia). Human SPINK1-overexpressing (OE) and SPINK1 short hairpin RNA (shRNA) lentiviral vectors were generated by Applied Biological Materials (ABM, Nanjing, China), and the Transwell system was purchased from Nest (Southborough, MA, USA). The following commercially available antibodies were used: anti-SPINK1 (Abnova, Taipei, Taiwan) and anti-Bcl-2, anti-Bcl-XL, anti-Bax, anti-Bad, and anti-caspase-3 (all from Wanlei Biotech, Shenyang, China). The main chemicals or reagents used in this study were as follows: 5-fluoruracil (5-FU) (Shanghai Pharmaceutical Company, Shanghai, China), Lipo2000 (Invitrogen, Carlsbad, CA, USA), Cell Counting Kit-8 (CCK-8) (Xian Baiying Biotechnology Inc., Xian, China), puromycin (Solarbio, Beijing, China), and polybrene (Maokang Biotech, Shanghai, China).

The MRC-5 and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sangon Biotech, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, Beijing, China) and cultured in 5% CO₂ at 37 °C. The A549 cells were stably upregulated and downregulated for Cathepsin K expression using the Cathepsin K plasmid and siRNA (Genepharma, Shanghai, China), respectively. DNA transfection was performed using Lipofectamine-3000 (Thermo Fisher, Waltham, MA, USA), and siRNA was transfected at a final concentration of 50 nM using Lipofectamin-3000.