Figure 3.

NTN 4 induces cell migration in neuroblastoma cell line, SK-N-SH, through NEO1/Lamimin γ1 interaction (a) Representative images of transwell assays using rhNTN4 and mLM-111 as adhesion molecules in SK-N-SH cells shSCR and shNEO1. Briefly, transwell assays were performed in chambers with an 8μm-pore membrane. Chambers were pre-treated with PBS, 10 μg/ml mLM-111 and/or 2 μg/ml rhNTN4 and placed on the underside of the membrane for 12h before performing the assay. As a chemotactic stimulus DMEM medium supplemented with 5% FBS was used. Cell migration was allowed for 2 h and analysis was performed as described in the material and methods. Data were normalized with respect to shSCR cells (PBS condition). Scale bar 100 μm. (b) Quantification of transwell assays obtained in C. Migrated cells were counted using inverted microscopy at 100x magnification. Five fields per condition were counted and data represent the average from three independent experiments. Data was normalized to shSCR cells (PBS condition). Mann Whitney t-test *p < 0,05 shSCR PBS versus shSCR mLM-111, rhNTN4 or both, α p < 0,05 shSCR versus shNEO1 in the same cell adhesion stimulus. (c), (d) Representative western blots of protein co-immunoprecipitation assays used to evaluate interaction between NEO1 and LMγ1 in SK-N-SH cells. Cells were lysed and incubated using specific antibodies against either LMγ1 (c) or NEO1 (d) followed by western blot against NEO1 and LMγ1.