Fig. 2

Vps26a knockdown prolongs the maintenance of stemness under ND conditions in P19 ECCs. a, b The effect of Vps26a knockdown on the expression of ESC stemness genes were determined by semi-qPCR (a) and western blot (b) analyses of Oct3/4 and Nanog using shCTL- and shVps26a-ECCs. c Morphological changes of shCTL (shC)- and shVps26a (shV)-ECCs during retinoic acid-induced neurogenesis (RA-ND) for the indicated time periods. The yellow arrowheads indicate cells with differentiated morphologies. d–f Expression kinetics of ESC stemness and neuronal markers examined by semi-qPCR (d), qPCR (e), and western blotting (f) analyses using shC- and shV-ECCs differentiated for the indicated time periods. Error bars indicate the means ± SD (n = 3). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 compared with the shC-ECCs from each day during ND. f–i Immunoblotting quantification of (f). Quantification of the fluorescence intensity was performed using ImageJ software (n = 3 for each group). Error bars are ± SD. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 vs. shCTL ND 0. g Double-label immunocytochemical analysis of Oct3/4 (green) and Tubb3 (red) during ND for 48 h. DAPI staining data are shown as insets to the merged images. Scale bar, 50 μm