Fig. 7: Arhgap17 knockdown or Rac1 activation rescues the GNP proliferation defect caused by Wdr4 deletion.

A, D, I Quantitative data measuring proliferation in GNPs isolated from P7 Wdr4 A-cKO mice and infected with lentiviruses carrying GFP and shRNA against Arhgap17 or control (A), treated with Rac1 activator ML099 or DMSO (D), or infected with lentiviruses carrying GFP and Wdr4 WT or mutants (I) at DIV 0. The capability of Arhgap17 shRNAs to knockdown the expression of Arhgap17 is shown in Fig. S7. The total cell numbers at DIV 0 and DIV 3 were counted. Data were from 3 (A, I) or 6 (D) cerebella in each group and analyzed using one-way ANOVA post hoc Holm-Šídák’s test (A), two-tailed unpaired Student’s t-test with Welch’s correction (unequal variances, D), or one-way ANOVA post hoc Dunnett’s test (I). p = 0.012 in shArhgap17#1 v.s. shCtrl, and 0.0376 in shArhgap17#2 v.s. shCtrl (A), p = 0.0082 in (D), p = 0.0001 in Wdr4 WT, >0.9999 in Wdr4 D166A, and 0.5937 in Wdr4 R172Q, compared to the control group (I). B, C, J, K Representative confocal images (B, J) and quantitative data (C, K) for measuring cell cycle exit in P7 Wdr4 A-cKO GNP cultures with Arhgap17 knockdown (B, C) or overexpression of WT or mutant Wdr4 (J, K). The GNPs were isolated, treated with EdU for 2 h, infected with lentiviruses carrying GFP and shRNA or cDNA at DIV 0, and harvested for staining analysis at DIV 3. Data were from 3 cerebella in each group and analyzed using one-way ANOVA post hoc Dunnett’s test. p = 0.0055 in shArhgap17#1 v.s. shCtrl, and 0.0256 in shArhgap17#2 v.s. shCtrl (C), p < 0.0001 in Wdr4 WT, >0.9999 in Wdr4 D166A, and 0.3568 in Wdr4 R172, compared to the control group (K). E, F Representative confocal images (E) and quantitative data (F) for measuring cell cycle exit in P7 Wdr4 A-cKO; Ai14 GNP cultures treated with ML099 or DMSO at DIV 0 and harvested 3 days later. The GNP cultures were treated with EdU for 2 h before ML099 treatment. Data were from 3 cerebella in each group and analyzed using two-tailed unpaired Student’s t-test without Welch’s correction (equal variances), p = 0.0052. G, H Representative confocal images (G) and quantitative data (H) from the EGL of lobule V–VI measuring the number of Ki67⁺ proliferating GNPs in the P7 Wdr4 A-cKO or control cerebella. The Wdr4 A-cKO and control mice were injected once with DMSO or ML099 (20 mg/kg) at P3, and harvested 4 days later. Data were from 5 cerebella in each group and analyzed using one-way ANOVA post hoc Turkey’s test, p < 0.0001 in (H, A-cKO v.s. Ctrl with DMSO), and =0.0439 in (H, ML099 v.s. DMSO in A-cKO). See also Fig. S8. Scale bars are 25 μm in (B, E, J) and 50 μm in (G). Data are represented as individual points and mean, or mean ± S.E.M.; *p < 0.05, ***p < 0.0005; n.s., non-significant. L Western blot analysis for Wdr4 expression in GNPs infected with lentiviruses carrying Wdr4 WT or mutants at DIV 0 and harvested at DIV 3, showing comparable expression levels among WT and mutants. M Western blot analysis for Arhgap17 expression in N2a cells overexpressing Wdr4 WT or mutants. The western blot results were quantified using ImageJ software. The protein levels were normalized first to the Gapdh protein level in each group and then to the corresponding control groups, and expressed as fold changes at the bottom. All western blot analyses were done at least twice.