Figure 5

Depletion of Usp9x increases Wnt target gene expression. (A,B) qRT-PCR analysis of RNA isolated from E12.5, E14.5 and E16.5 neocortices of Usp9x ^+/Y and Usp9x ^−/Y (n = 3). Canonical Wnt signalling target genes Ccnd1 (A) and Axin2 (B) were up regulated in the Usp9x ^−/Y neocortices compared to Usp9x ^+/Y in all tested embryonic stages. (C) TCF-TOPFlash reporter activity was significantly increased in USP9X siRNA transfected HEK293 compared to scrambled nonsense siRNA transfected cells. Addition of Wnt3a increased TOPFlash reporter activity in scrambled siRNA cells to a level similar to Usp9x siRNA treated cells (n = 3). Addition of the Wnt antagonist DKK1, significantly reduced TOPFlash reporter activity only in the Scramble siRNA HEK293 cells (n = 3). (D) Increased total β-catenin protein levels in USP9X-depleted ReNcell VM. (E,F) Expression of canonical Wnt signaling target genes, CCND1 and AXIN2 was significantly increased in USP9X-depleted ReNcell VM cells. (G) Inhibition of USP9X deubiquitylating activity in ReNcell VM cells by WP1130 increased total β-catenin levels to a level similar to the proteasome inhibitor epoxomicin. DMSO was used as the vehicle control. (H) Quantitative RT-PCR analysis of CCND1 gene expression identified significant increases in USP9X-depleted ReNcell VMs compared with those expose to Scrambled shRNA. Treatment with Wnt3a increased CCND1 expression in untreated (WntWT) as well as scrambled and Usp9x shRNA treated ReNcell VMs. Statistical significance was assessed by one-way ANOVA, followed by Tukey’s post-test. ***p < 0.001; **p < 0.01; *p < 0.05.