Transwell migration assays

Tranwell assays were completed using a chamber within an 8μm-pore polycarbonate membrane (Corning). As a haptotactic stimulus, 2μg/μl fibronectin was used (Sigma Aldrich), placed on the bottom of the membrane 12h before performing the assay. As a chemotactic stimulus, different concentrations of human recombinant Netrin-4 (R&D Systems) diluted in DMEM (without FBS) were used; the concentrations are indicated in Figure ​Figure4.4. Briefly, for SK-N-SH, 50,000 shNEO1, shNTN4, and shSCR cells were placed in the upper chamber; the bottom chamber contained NTN4 diluted in DMEM. The cells were incubated for 4h, fixed, and stained using Crystal violet 100% diluted in methanol in a solution 1:5 of NaCl 0,15M. For LAN-1 100,000 shNEO1, shNTN4, and shSCR cells were placed in the upper chamber; the bottom chamber contained 100 ng/ml Netrin-4 diluted in DMEM. The cells were incubated for 6h, fixed, and stained same as SK-N-SH cells.

To determine if NEO1-overexpressed cells migrate more in transwell assay than control cells in the presence of a Netrin-4 stimulus (100ng/mL), SK-N-SH cells overexpressing EV (empty vector), NEO1GFP, or NEO1ICDGFP were used. The results were normalized according to condition without Netrin-4 for each experiment.