Transwell Migration and Invasion Assay

Transwell migration and invasion assays were conducted as previously described [13 (link)]. For invasion assay, the upper compartment of the transwell chamber (8 μm pore size, 24 wells, Millipore, Billerica, USA) were precoated with 10 μL of diluted Matrigel (Matrigel and RPMI 1640, 1:3, v/v, BD Biosciences, Sparks, USA). Cells were suspended in serum-free medium and seeded into upper chambers at a density of 3 × 10⁴ and 5 × 10⁴ cells for migration and invasion assays, respectively. To examine the effect of signaling agonists and inhibitors on the invasiveness of GC cells, the Matrigel-transwell system were cultured with or without IFNγ (50 ng/mL, R&D Systems, Minnesota, USA), STAT1 siRNA (40 nmol/L), JAK inhibitor I (15 ng/mL, Santa Cruz Biotechnology), ZEB1 siRNA (50 nmol/L), and IRF1 siRNA (50 nmol/L). RPMI-1640 medium containing 10% FBS and indicated inhibitors or agonists were added to lower chamber (600 μL/well). After incubation for 24 h, the non-invasive cells on the top surface of upper chamber were carefully removed with a cotton swab. Cells were fixed, stained and counted. Each experiment was repeated three times.