Fig. 1: CUL4B deficiency results in premature cell cycle exit and precocious neuron differentiation of NPCs in 2D culture.

A The mRNA levels of CUL4B in different iPSC lines were quantified by qRT-PCR. N = 3. B Western blots showing CUL4B protein levels in different iPSC lines. The number below each CUL4B band is the normalized ratio between the intensity of CUL4B band and the intensity of GAPDH band from the same sample. C The upper is the schematic showing neural differentiation strategy for iPSCs. The lower is the immunostaining for the NPC markers PAX6, NESTIN, SOX2, N-Cadherin, SOX1, the forebrain marker FOXG1 and CUL4B in NPCs on day 17. Scale bar, 50 μm. D The representative images of NPCs stained with SOX2 and Ki67 in day-16 (D16) and day-25 (D25) cell cultures and quantifications of the percentage of Ki67⁺ cells among SOX2⁺ cells. Scale bar, 50 μm. N = 12. E The mRNA levels of CUL4B, PAX6, TUJ1 and DCX in different cell lines. N = 3. F Ki67 staining (green) of NPCs at 48 h after EdU labeling (red). Scale bar, 50μm. The bar graph shows the percentage of EdU⁺ Ki67⁻ cells in all EdU⁺ cells. N = 12-14. G DCX staining (green) of NPCs at 48 h after EdU labeling (red). Scale bar, 50 μm. The bar graph shows the percentage of EdU⁺ DCX⁺ cells in all EdU⁺ cells. N = 12. The statistical significance was determined using one-way ANOVA with Tukey test. *: P < 0.05; **: P < 0.01; ***: P < 0.001. ns: no significance.