The m5C reader Ybx1 regulates embryonic cortical neurogenesis by promoting progenitor cell cycle progression
Fig 6
Knockdown of Ybx1 target mRNAs impairs the proliferation and differentiation of cortical progenitor cells.
(A) Representative images of neurospheres formed after siRNA-mediated knockdown of Ccnd2, eEF1g, Rpsa, Rps5, or Uhmk1 in cultured E13.5 neural stem cells (NSCs). Scale bar, 50 μm. (B and C) Quantification of the sizes (B) and numbers (C) of the neurospheres after siRNA-mediated knockdown of Ybx1 target mRNAs show in (A). At least 3 replicates were performed. All statistical data are presented as box and whisker plots: in B, siCcnd2 (n = 55 neurospheres) vs. siCtrl (n = 51 neurospheres), ****p = 5.87E−06; sieEF1g (n = 52 neurospheres) vs. siCtrl, ****p = 5.46E−07; siRpsa (n = 57 neurospheres) vs. siCtrl, ****p = 1.57E−07; siRps5 (n = 49 neurospheres) vs. siCtrl, ****p = 6.12E−07; siUhmk1 (n = 45 neurospheres) vs. siCtrl, ****p = 9.87E−08; by one-way ANOVA followed by Tukey’s multiple comparison test; ns, not significant. (D) Immunostaining for GFP, EdU, and Ki67 on coronal sections of E16.5 mouse cortex after knockdown of Ybx1 targets in the cortex using IUE of a cocktail shRNA against all targets. Scale bar, 50 μm. (E and F) Quantification of percentage of EdU+GFP+/GFP+ (E) and Ki67-EdU+/EdU+GFP+ (F) shown in (D). At least 3 embryos were analyzed for each condition. Data are presented as box and whisker plots: in E, shCtrl (n = 23 confocal fields) vs. shCocktail (n = 27 confocal fields), ***p = 2.10E−04; in F, shCtrl (n = 23 confocal fields) vs. shCocktail (n = 27 confocal fields), ***p = 6.86E−04; by unpaired Student t test (E, F). The data underlying all the graphs shown in the figure are included in S1 Data.