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. Author manuscript; available in PMC: 2013 Dec 7.
Published in final edited form as: Cell Rep. 2013 Oct 24;5(3):10.1016/j.celrep.2013.09.034. doi: 10.1016/j.celrep.2013.09.034

Figure 1. Smek1 regulates neuronal differentiation in the early phase of NPC differentiation.

Figure 1

(A, B) Smek1 expression in mouse forebrain cortex. Coronal sections from forebrain cortex of E11.5 (A) and E14.5 (B) mouse embryos were immunostained with Smek1 and Nestin (an NPC marker) or TUJ1 (a marker of immature neurons) antibodies. Hoechst dye served as a counterstain. Smek1 proteins are detected mainly in Nestin-positive cells of the VZ of E11.5 cortex (A) and in TUJ1-positive cells of the CP at E14.5 (B). Notably, Smek1 is also observed in cells at the ventricular surface (box) and in TUJ1-positive cells (arrows) of the VZ of E14.5 cortex. Scale bar, 100 μm. PP, preplate; VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate.

(C) Quantitative analysis of Pax6 and Tbr1-positive cells in E14.5 Smek+/+ and Smekgt/gt forebrain cortices. Fold difference was evaluated by normalization of the average number of cells positive for the indicated antibodies per coronal sections against wild-type mice. Error bars represent the mean ± S.D. of three independent experiments. **p < 0.05.

(D) Smek1 depletion inhibits neuronal differentiation. NPCs expressing Smek1 shRNA or control shRNA under control of doxycycline-inducible promoter were cultured in differentiating condition medium containing doxycycline for 6 days and then subjected to immunostaining for TUJ1, MAP2 (a marker of mature neurons), or Smek1. Percentages of TUJ1- and MAP2-positive cells per total cells are shown in the bottom panel. Scale bar, 100 μm.

(E) Smek1 depletion decreases neuronal differentiation. Two days prior to differentiation, expression of Smek1 shRNA or control shRNA was induced by addition of doxycycline. Cells were then cultured under differentiating conditions for indicated times and then immunostained with TUJ1, Nestin, Pax6, or Ki67 antibodies. Scale bar, 100 μm.

(F) Percentages of neuronal cells or undifferentiated NPCs were evaluated by counting TUJ1-positive cells, Nestin/Ki67 double-positive cells, or Pax6-positive cells per total cells. Error bars represent the mean ± S.D. of four independent experiments; each assay was performed in duplicate. *p < 0.001; **p < 0.05; n.s., not significant.