RA stimulates accelerated neural specification. 46C ESCs were cultured under monolayer differentiation conditions with RA (N2B27 + 10-8 M RA) or without RA (N2B27 + 0 or N2B27 + ethanol). A, B, Typical FACS profile of Sox1GFP expression at day 2. M1 is the gate used throughout to quantify the proportion Sox1GFP+ cells; C-E, Proportion of Sox1GFP+ cells at various time points (average of triplicates). *, p < 0.05; **, p < 0.01. D, Treatment of adherent cultures with RA after the appearance of the first Sox1GFP+ cells; E, Treatment of purified Sox1GFP+ cells with RA; F, Ratios of clones containing GFP+ cells (GFP+) or without GFP+ cells (GFP-) when cultured at clonal density. Under each culture condition, 28 clones were observed and counted. #, significant difference in this ratio from controls (p < 0.001, using contingency table analysis); G, Western blot analysis of Oct4 at day 4 under monolayer cultures; H, RT-PCR for the ESCs marker Oct4 during monolayer differentiation. To normalize their expression to the amount of cDNA present in the sample, the cDNA for endogenous GAPDH was amplified; I, Q-PCR for the ESCs marker Oct4 during monolayer differentiation.