Matrigel invasion assay

Invasion assays were performed by using the same transwell chamber with growth factor-reduced Matrigel. Briefly, 1 × 10⁵ cells infected with LV- NC or LV-LAMA4-shRNA3 per well were seeded onto Matrigel-coated inserts and allowed to invade for 48 h. Cells remaining above the insert membrane were removed with a cotton swab, and cells that invaded through the Matrigel were fixed in 25% methanol. After washing in cold 1 × PBS, the cells were stained with 0.1% crystal violet in 25% methanol. The inserts were washed three times with 1 × PBS and then air-dried. The numbers of invaded cells on the representative sections were counted using an inverted microscope (IX71; Olympus) at 10× magnification. Five fields were counted per filter in each group; the number of invaded cells for each sample represents the average of triplicate wells over three experiments.