Fig. 6: Hedgehog pathway activity in single and double mutant cells and its modulation by NDE1.

a Summary data of Hedgehog pathway activity normalized to wild type, after transfection of wild type, Fbxw7KO, Nde1KO, and Fbxw7Nde1KO C3H10T1/2 cells with a Gli-reporter construct. Data are presented as means ± SEM from three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, ***p < 0.001, ****p < 0.0001. b Summary data of Wnt pathway activity normalized to wild type, after transfection of wild type, Fbxw7KO, Nde1KO, and Fbxw7Nde1KO C3H10T1/2 cells with a βcatenin-reporter construct. Data are presented as means ± SEM from four independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test, **p < 0.01, ****p < 0.0001. c mRNA levels of Wnt9a in wild type, Fbxw7KO, Nde1KO, and Fbxw7Nde1KO C3H10T1/2 cells (n = 3 experiments). Data are presented as means ± SEM. One-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, ***p < 0.001. d, e Summary data of Hedgehog pathway activity, normalized to untransfected, after transfection of C3H10T1/2 (d) or HEK293T cells (e) with a Gli-reporter construct and the indicated constructs (n = 3 experiments). Expression of the indicated constructs is shown at the bottom of each graph. Data are presented as means ± SEM. One-way ANOVA with Sidak’s multiple comparisons test, *p < 0.05, ****p < 0.0001. f Physical interaction of GLI2-myc and NDE1-FLAG constructs in HEK293T cells. g, h Representative images of wild type and Nde1KO C3H10T1/2 cells transiently transfected with a GLI2ΔN-myc construct encoding an uncleavable form of GLI2 (active GLI2). Cells were stained for myc-tag (green, g). Summary data from of the nuclear:cytosol (N:C) ratio of GLI2ΔN-myc in the indicated cells (h). Data were pooled from three independent experiments and presented as means ± SEM. Student’s t-test, ****p < 0.0001.