Fig. 3

CD271 overexpression promotes cSCC differentiation a CD271 and mock spheroids were fixed with 4% PFA and embedded in paraffin. Spheroid histology was evaluated by Hematoxylin and Eosin (H&E) staining (scale bar = 50 μm) and b the number of nuclei was measured by ImageJ software. c Density, Weight, and Diameter of SCC13-Spheroids measured by W8™. A minimum of 10 single spheroids was analyzed for each test condition and values were extrapolated from at least 10 repetitions. d The expression of CD271, TrkA, KRT1 and Ki67 were evaluated in mock vs CD271-overexpressing SCC13-spheroids by immunofluorescence. Nuclei were stained with DAPI. e Left panel: Viability and apoptosis were measured in CD271 vs mock spheroids by LIVE/DEAD® assay (Calcein: Green, Ethidium Bromide Red). Right panel: percentage of Ethidium Bromide positive cells determined by ImageJ software analysis of micrographs. Scale bar = 50 μm f CD271 and mock spheroids implanted into collagen I and dermal fibroblast matrix and monitored for 2 weeks (scale bar = 100 μm). % of fragmentation and invasion area was determined by ImageJ software analysis. g H&E and CD271, KRT10, and KRT15 expression evaluated by immunofluorescence of mock and CD271-overexpressing SCC13-derived skin reconstruct. Nuclei were stained with DAPI. Statistical analysis was performed using the two-way ANOVA and multiparametric T-test. *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****p < 0.0001. Scale bar = 50 µm