Lentiviral Knockdown of COL4A1 in Gastric Cancer

The production of lentivirus carrying short hairpin RNA (shRNA) was constructed as previously reported³⁵ (link) to knock down the expression of COL4A1 in GC cells. For transduction, GC cells were infected for 24 h with 10 μL of virus suspension containing 8 μg/mL Polybrene (Sigma, VT, USA). The cell functions were tested using a Transwell Boyden chamber (Costar, USA) with polycarbonate membranes (8-μm pore size) either coated with 10 μg of Matrigel (BD Biosciences, Bedford, MA, USA) per well (for invasion assays) or left uncoated (for migration assays) in serum-free medium containing 10 g/L bovine serum albumin on the bottom of the upper compartment. The cells were suspended in serum-free DMEM medium at a total amount of 5 × 10⁵ cells; simultaneously, 0.5 mL of DMEM with 10% FBS was added to the lower compartment served as a chemoattractant. Non-invading cells were removed with cotton swabs after 36 h. Invaded cells were fixed with 90% ethanol for 15 min at room temperature and stained with 0.1% crystal violet solution. Images of migrated and invaded cells were captured by a photomicroscope and quantified by blind counting with five fields per chamber.