Induced release of monomeric laminin-γ2 upon transient laminin-α3 knockdown in SW620 cells and its impact on cell migration. (A) Northern blot analysis of LAMA3 knockdown in SW620 cells. RNAs were prepared from SW620 cells transiently transfected with LAMA3 siRNA and incubated with TNFα for 24 h. Quantification of LAMA3 messages normalized for GAPDH is indicated. (B) Western blot analysis of laminin-γ2 expression upon transient knockdown of LAMA3. SW620 cells transiently transfected with LAMA3 siRNA or non-targeting siRNA were shifted to serum-free cultures 48 h after transfection and were incubated with TNFα for another 48 h. Proteins from conditioned media (16 μg/lane) were probed with a laminin-γ2-specific antibody (2140). Quantification of the monomer and of the heterotrimer normalized for transferrin is indicated. (C) Migration of SW620 cells as analyzed in a transwell migration assay. SW620 cells transfected with LAMA3 siRNA or non-targeting siRNA were plated in a transwell chamber 24 h after transfection. TNFα was added one day later. Migrating cells were quantified after three days using Cell Titer Glo assay (Promega). Bars show the mean value of four experiments with the standard error of the mean. Statistical analysis was carried out by t test (one-tailed, GraphPad Prism 4.00).