Fig. 3: MYCN activation induces characteristics of a tumorigenic phenotype in differentiating NCSC

a A MTS/PMS assay was used to measure cell proliferation. MYCN+ cells had more than two times proliferation rates compared to MYCN- (****p < 0.0001). Representative microscopy images (20X) of MYCN- and MYCN+ are shown. b Anchorage-independent growth was assessed by colony formation from single cells in soft agar. MYCN- and MYCN+ were each combined in a soft agar mix and separately plated for 1 month. Soft agar plates were stained with crystal violet. Representative light microscopy images for MYCN+ at 10× and 20× are shown. Colonies were counted manually and by ImageJ software. MYCN+ formed over 50 colonies while MYCN- and controls had no colony formation. (***p < 0.001) c: To measure migration, MYCN- and MYCN+ cells were seeded in wells of a transwell plate containing 1 µm pores. Cells that migrated through the transwell pores were stained with crystal violet and quantified. Representative light microscopy images of MYCN- and MYCN+ stained with crystal violet are shown. MYCN+ migrated significantly compared to the rate of MYCN- controls (****p < 0.0001). d Invasiveness was similarly assessed by adding a matrigel layer to the transwell porous system. MYCN+ cells were significantly more invasive than MYCN- and controls (****p < 0.0001). e and f Protein expression levels of NBL tumor diagnostic markers (TH, Phox2b, and TRKB) were analyzed in MYCN- and MYCN+ cells at day D14 using immunocytochemistry and immunoblotting. While MYCN- had minimal NBL marker expression at D14, MYCN+ had high expression of all three NBL markers tested. Bars represent mean ± 2SD. Statistical analyses were performed with ANOVA