Impact of AdOx pretreatment on RA-induced differentiation of P19 cells. A. Schematic of the effect of AdOx pre-treatment on RA-induced P19 cells. B. Microscopic images showing the impact of AdOx on RA-induced P19 cells. P19 cells were pre-treated with AdOx (+) or without (-) for 1 day (0 d) followed by RA treatment for 4 days (4 d) and then cultured in RA-free medium for another 4 days (8 d). C. Immunostaining for Tuj1 (green) in RA-induced P19 cells pre-treated with AdOx (+) or without (-). D. Quantification of Tuj1-positive P19 cells induced by RA and pretreated with AdOx (AdOx) or without (control). E. Impact of AdOx pretreatment on the expression of Tuj1 and β-actin in RA-induced P19 cells as shown in Western blots and described as in D. F. Impact of AdOx concentration on the expression of Tuj1 in P19 cells induced with RA (+) or without (-) as shown in Western blots. GAPDH and Hsp90α are the loading controls. Numbers indicated quantification of the Tuj1 western signal relative to GAPDH. Digits at the bottom of the first row indicates the relative darkness of each band compared with the control lane set as 1.0. G. RT-qPCR analyses of ngn1, mash1, and neuroD mRNA expression in P19 cells pre-treated with AdOx (AdOx) or without (Control) for 1 day (RA 0 d) and then induced with RA (RA 2 d). Each bar in the histograms of this figure represents a mean value with S.D. from three independent experiments (mean ± SD). Asterisks represent significance in statistics: (*) as P < 0.05, (**) for P < 0.01 and (***) for P < 0.001.