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. 2020 Aug 6;9:e57381. doi: 10.7554/eLife.57381

Figure 6. Depletion of Dyrk2 induces abnormal ciliary trafficking of endogenous Hh components.

Ciliary localization of endogenous SMO, GLI2, and GLI3 in wild-type and Dyrk2-/- MEFs in the absence or presence of 100 nM SAG. Primary cilia were immuno-stained for SMO (A), GLI2 (C), or GLI3 (E) with ARL13B and gamma-tubulin (white) antibodies. Nuclei were stained with DAPI (blue). The percentage of cells with SMO (B) at the cilia or foci of GLI2 (D) or GLI3 (F) at the cilia tips was determined. Data are presented as the means ± SEM (n = 3 biological replicates for each condition;>110 cells were scored for each experiment). The statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. (*) p<0.05, (**) p<0.01. Scale bars, 5 µm.

Figure 6—source data 1. Source data for Figure 6B,D and F.

Figure 6.

Figure 6—figure supplement 1. Depletion of Dyrk2 induces abnormal ciliary trafficking of endogenous GLI2 and GLI3 in vivo.

Figure 6—figure supplement 1.

Immunohistochemistry for GLI2 and GLI3 in wild-type and Dyrk2-/- mesenchymal cells in the craniofacial region at E10.5 tissues. Primary cilia were immuno-stained for GLI2 (A) or GLI3 (B) with ARL13B and gamma-tubulin (white) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 5 µm.
Figure 6—figure supplement 2. Immunocytochemistry of endogenous SuFu and IFTs.

Figure 6—figure supplement 2.

(A) Ciliary localization of endogenous SuFu in wild-type and Dyrk2-/- MEFs in the absence or presence of 100 nM SAG. Primary cilia were immunostained for SuFu with ARL13B and gamma-tubulin (white) antibodies. (B–D) Ciliary localization of endogenous IFTs in wild-type and Dyrk2-/- MEFs. Primary cilia were immuno-stained for IFT140 (B), IFT81 (C), or IFT88 (D) with acetylated-tubulin and gamma-tubulin (white) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 5 µm.
Figure 6—figure supplement 3. Effects of rapamycin treatment on cilia.

Figure 6—figure supplement 3.

(A) Phosphorylated protein levels of S6K and 4EBP in wild-type and Dyrk2-/- MEFs were measured by immunoblotting. GAPDH serves as a loading control. (B) Primary cilia in wild-type and Dyrk2-/- MEFs treated with vehicle (DMSO) or 0.5 µM rapamycin for 24 hr were immunostained with acetylated-tubulin (red) and gamma-tubulin (green) antibodies. Nuclei were stained with DAPI (blue). Scale bars, 5 µm. (C, D) Measurements of cilia length in wild-type and Dyrk2-/- MEFs treated with vehicle (DMSO) or 0.5 µM rapamycin using acetylated-tubulin as a cilia axoneme marker. Cilia lengths are presented as pooled from three MEFs derived from independent embryos of each genotype (C) and the average of each MEF (D). Data are presented as the means ± SEM (n = 3 biological replicates per condition). The statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test.
Figure 6—figure supplement 3—source data 1. Source data for Figure 6—figure supplement 3C–D.
Figure 6—figure supplement 4. Protein levels of CP110 and KATANIN p60 in Dyrk2-/- MEFs.

Figure 6—figure supplement 4.

Protein levels of CP110 and KATANIN p60 in wild-type and Dyrk2-/- were measured by immune-blotting. GAPDH serves as a loading control.