Fig. 4. WDR81 is required for endosomal trafficking of EGFR.

a Expression of control shRNA and shRNA-mediated knockdown constructs for WDR81, WDR91 and p62, together with pCAG-GFP. Plasmids were delivered by in utero electroporation at E13.5 and analysis was performed at E16.5. Ventricular Zone and Sub-Ventricular Zone (VZ + SVZ), Intermediate Zone (IZ) and Cortical Plate (CP) were identified based on DAPI staining. b Quantification of electroporated cell distribution reveals major accumulation in the IZ following WDR81 and WDR91 knockdown (VZ + SVZ, WDR91 shRNA p = 0.0025; IZ, WDR81 shRNA p = 0.0001, WDR91 shRNA p = 0.067; CP, WDR81 shRNA p = 0.0003, WDR91 shRNA p = 0.0234) (n = 3–4 independent brains per condition). c Ventricular zone of E14.5 WT and WDR81^−/− mice cortices stained for EEA1 and Actin. d Control and WDR81 patient fibroblasts stained for EEA1. e Quantification of individual EEA1+ early endosomes in WT and WDR81^−/− VZ in 200 × 100 μm crops reveals increased size in mutant brains (p < 0.0001) (n = 3 independent experiments). f Quantification of individual EEA1+ early endosomes in control and WDR81 patient fibroblasts reveals increased size in mutant cells (For all control-patient couples p < 0.0001) (n = 3 independent experiments). g EGF⁵⁵⁵ uptake assay in control and WDR81 patient fibroblasts, and stained for EEA1. h Quantification of EGF⁵⁵⁵ and EEA1 colocalization during EGF⁵⁵⁵ uptake reveals prolonged colocalization between EGF and early endosomes in WDR81 patient cells (At T15, C1 vs P2 p = 0.0158; C2 vs P1 p < 0.0001; C2 vs P2 p < 0.0001. At T30, C2 vs P1 p = 0.0149, C2 vs P2 p = 0.0033. At T60, C1 vs P1 p < 0.0001; C1 vs P2 p = 0.001; C2 vs P1 p = 0.0028. At T120, C1 vs P1 p = 0.0008; C1 vs P2 p = 0.0011; C2 vs P1 p = 0.0072; C2 vs P2 p = 0.0148. At T360, C1 vs P1 p = 0.0349; C2 vs P1 p < 0.0001. C2 vs P2 p = 0.0001) (n = 7 independent experiments). All experiments were performed at least three independent times. All data are expressed as mean ± standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-tailed unpaired t test (A & H) and Mann–Whitney tests (E & F).