Figure 4

Analysis of migration, invasion, and anchorage-independent growth in DAOY and SHH-NPD cells. A, In vitro scratch assay in which monolayers of SHH-NPD and DAOY cells, previously infected with HIVZsGreen lentivirus for stable expression of Arnt, were scratched to create a linear wound and photographed 0 and 10 hours later. The average rate of cell migration (μm/hr) across the wound gap was determined by measuring the distance between opposing cell fronts. Fluorescence microscopy verified expression of ZsGreen-tagged Arnt and Gdi2 in the migrating cells. Bar graphs show the mean rate of cell migration (μm/hr) across the gap. B, Matrigel chemoinvasion assay in which SHH-NPD and DAOY cells expressing lentivirus-transferred Arnt and Gdi2 or empty virus (control) were placed in the upper wells of BD Biocoat Matrigel chambers and the cells invading through the matrigel to the lower chamber (arrows) were counted. Bar graphs show the mean number of invading cells/microscope field in two separate experiments. C, Soft agar colony forming assay. Bar graphs show the mean number of colonies (>38 μm) per well (35-mm diameter) 14 days after plating in 0.3% agar. Error bars, SD. Scale bar, 400 μm. Statistical significance, compared with control, indicated by asterisks (P < 0.05) or “ns” (not significant).