Transwell system

Cell suspensions (2 × 10⁵ cells/mL) of CC cells were prepared utilizing serum-free medium. A total of 0.2 mL cell suspension was added to the upper compartment of the Transwell system (Nest, Wuxi, China) while the lower compartment was filled with the medium containing 10% FBS. After the cells were placed in the incubator for 12 h, the migrated and invaded cells were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet solution. Under a microscope, five visual fields were selected in each group for counting the cell number. For the invasion assay, the bottom of the Transwell system was coated with a layer of Matrigel (30 µg/well; BD, San Jose, CA, USA) before the inoculation of the cells.

Migration and invasion assays were performed using the Transwell system (Nest, 723,001). For the 48 h migration assay, MDA-MB-231 and MDA-MB-468 cells (1 × 10⁵) were seeded in small chambers (Nest, 723,001) with a serum-free medium, and a 700 mL medium with 20% FBS was added to the bottom wells. Likewise, for invasion assay, 1 × 10⁵ cells were seeded in Matrigel-coated chambers with serum-free medium and cultured for 48 h along with L15 supplemented with 20% FBS in the bottom wells. The images were captured by microscope (Nikon, Japan).

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).