Figure 5

The effect of reduced Npas4 expression on Nestin expression during neural differentiation of mESCs. (A) Immunocytochemical analysis of Nestin expression (green) during neural differentiation. Representative images of cultures are shown for each mESC line at Day 6 to 9 of N2B27 differentiation. Cells were counterstained with DAPI to visualize nuclei (blue). Scale bar = 100 μm. (B) Quantification of Nestin expression in Npas4 knockdown and control mESC lines at various stages of neural differentiation. For each field, the overall fluorescence intensity was calculated for each color channel and from this the ratio of Nestin:DAPI fluorescence was determined. Means and standard deviations of three independent experiments are shown (n = 3). Over the course of the differentiation period, there was a statistically significant difference in the Nestin:DAPI ratio between the Empty vector control line and each of the Npas4 knockdown lines (Empty versus KD1, **P <0.01; Empty versus KD2, **P <0.01) when analyzed using a two-way ANOVA. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; mESC, mouse embryonic stem cells.