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Fig. 2. Adhesive activity of NcadER activated or not by 4OHT. (A) Aggregation of L cells, LN cells, and LNER cells with or without 4OHT treatment. Trypsin harvested cells were pipetted into a single cell suspension and identical numbers placed in hanging drop cultures, with LNER cells treated with or without 100 nM 4OHT. After 24 hours the cells were collected, triturated and photographed under phase-contrast microscopy. Non-transfected parent L cells (L) failed to aggregate, whereas LN cells formed tightly compacted aggregates. LNER cells without 4OHT (LNER - 4OHT) formed large aggregates only partly dispersed by pipetting and where individual cells were clearly discernable. LNER cells with 4OHT (LNER + 4OHT) formed tightly compacted aggregates similar to those formed by LN cells. Compacted aggregates were highly resistant to dispersion by trituration, individual cells were difficult to discern, and a phase-dense ring was observed around the aggregate (arrows). (B) Behavior of LNER monolayers with or without 4OHT. 50,000 cells were plated in 100 µl medium in wells of six-well culture dishes. After the cells attached (
5 hours), the dishes were gently flooded with 2 ml medium, with or without 100 nM 4OHT, and the cells were observed for 3 days. The cultures were photographed within (top panels), and outside of (bottom panels) the original plating area. The line defines the edge of the plating area, which is left of the line. LNER cells without 4OHT piled up on one another in the plating area, detached, and eventually seeded new colonies outside the plating area. In the presence of 4OHT the LNER cells stayed predominantly as a monolayer within the plating area, and seeded far fewer colonies outside this area, compared to cells without 4OHT.
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