ZEB1 was necessary for neuronal differentiation.
A, Western blotting showed ZEB1 protein levels at days 0, 15, and 35 of neural differentiation from hESCs with or without ZEB1 knockout. β-TUBULIN was the loading control. B, phase-contrast images of the indicated cells during different days of neural differentiation; n = 3. Scale bar: 50 μm. C, left, flow cytometry analysis with BrdU antibody using cells after 4 days attachment of spheres for neuronal differentiation (day 30). Right, statistical analysis of the left results; n = 3. D, left, cell apoptosis and death analysis with PE Annexin V/7-AAD Kit. Right, statistical analysis to show the percentage of viable cells (Negative) or cells in early apoptosis (Annexin V+, p < 0.001), in late apoptosis or already dead (7-AAD+ and Annexin V+ and 7-AAD+, p < 0.001); n = 3. ZEB1-KO, ZEB1 knockout. OE (ZEB1-KO), overexpress ZEB1 in ZEB1-knockout cells.