Figure 5

PAX3/7 expression in P19 cultures treated with dimethylsulfoxide (DMSO) and 10 nM retinoic acid (RA) is indicative of skeletal myogenesis and not neurogenesis. Panel I: P19[control] cells, undifferentiated in monolayer cultures (control) or differentiated with 1% DMSO, 1% DMSO+ 10 nM RA, or 1% DMSO + 1 μM RA for 9 days, were immunolabelled with anti-TuJ1 (green) to detect terminally differentiated neurons and stained with the nuclear marker Hoechst (blue). Panel II: P19[β-Cat/EnR] cells were treated as in Panel I. Panel III: Quantitative analysis of the percentage of Tuj1-positive cells was established, expressed as the percentage of the total cell number (n = 6-10). Panel IV: Immunofluorescent staining of PAX3/7 protein (green), committed neuronal precursors (doublecortin-positive, red), and terminally differentiated neurons (TuJ1-positive, blue) in triple-labelled P19[control] cultures treated with DMSO + 10 nM RA, demonstrating that PAX3/7-positive cells (arrow) are not neuronal precursors or neurons. Panel V: Quantitative analysis indicated that the overwhelming majority of PAX3/7-positive cells in all treatments were non-neuronal. Statistics were analysis of variance, post-hoc Bonferroni, *P < 0.05, Scale bars, 50 μm.