Figure 2

Upregulated expression of Foxm1 in P4 cerebellar NSCs and its effect on self-renewal. (A) RT-qPCR data showing differential expression in pre- and post-differentiation NSCs of mRNA for Foxm1 (*****P < 0.0001: 0.0000089) and the neuronal differentiation gene βIII-tubulin (*P < 0.05: 0.029) (Mann–Whitney U test). (B) Left: PCR assay of Foxm1 expression using isoform-specific primers. Agarose (2%) gel separation of the amplified product yielded two bands corresponding to Foxm1 isoforms 201 (400 bp) and 202 (300 bp). Full-length gel is presented in Supplementary Figure 8A. Right: Immunoblots showing endogenous levels of Foxm1, βIII-tubulin, and Actin (loading control) in three NSC cultures before and after differentiation. Full-length immunoblots are presented in Supplementary Figure 8B. (C) Left: Immunoblots showing endogenous levels of Foxm1 and Hsp70 (loading control) in NSCs transfected with siRNA against Foxm1 or non-targeting siRNA controls (siCtrl). Densitometric values appear below blots. Full-length immunoblots are presented in Supplementary Figure 8C. Middle: Representative bright-field images of neurospheres formed by NSCs transfected with siCtrl and siFoxm1. Scale bar: 100 μm. Right: RT-qPCR data showing mRNA levels of βIII-tubulin (*P < 0.05: 0.043) and S100b (*P < 0.05: 0.031) in NSCs transfected with siCtrl and siFoxm1. P values vs siCtrl. (Mann–Whitney U test). (D) Left: Neurosphere-formation capacity of NSCs transfected with siCtrl and siFoxm1 and treated with cyclopamine-KAAD (KAAD) to suppress endogenous Hedgehog signalling. Graphs show percentage of seeded cells that formed neurospheres. P values vs siCtrl. (**P < 0.01: 0.003, NS: Not significant 0.072) (Two-way ANOVA test). Right: Immunoblots showing endogenous levels of Foxm1 in NSCs transfected with siCtrl and siFoxm1 and treated with cyclopamine-KAAD (KAAD). Full-length immunoblots are presented in Supplementary Figure 9A. Bars in panels A, C, and D represent the mean (SD) of three independent experiments.