Fig. 1

EDA triggers PM translocation of its receptor EDAR. A Fluorescence images of EDAR-GFP (green) in EDAR-GFP⁺ cells are shown with treatment of EDA-A1 (EDA) conditioned medium (CM) at indicated time points. Cytoskeleton labeled with phalloidin (red). Nuclei counterstained with DAPI (blue). B Quantification shows the ratio of EDAR levels in plasma membrane (PM) to cytosolic compartment (Cyt) at each indicated time points in A. The ratio of PM/Cyt at 0 min was normalized to 1.0. In each condition, at least 10 random images (each image includes ≥ 3 cells) from 3 independent experiments were used for quantification. C Immunoblotting shows the successful expression of EDA-A1, EDA-A2 and TNF-α in CM. Medium collected from naïve HEK293 cells as control (CTL) CM. D Fluorescence images (green) show the cellular location of GFP, EDAR-GFP, XEDAR-GFP, or TNFR1-GFP treated with indicated CM containing EDA-A1, EDA-A2, or TNF-α. E Quantification shows the PM/Cyt ratio of EDAR, XEDAR and TNFR1 in cells with indicated treatment as in D. Data from at least 10 random images (each image includes ≥ 3 cells) in each condition from 3 independent experiments. F A diagram shows the workflow using EDAR-GFP⁺ cell culture and mouse eyelid organotypic culture with indicated EDA treatment. G–H Immunoblotting shows the levels of EDAR in Cyt (left) and PM (right) from EDAR-GFP⁺ cells G or EDA knockout (^−/−) eyelid cultures H. Actin or Na/K ATPase as loading control for Cyt or PM. Error bars indicate mean ± SD from at least 30 cells from 3 independent experiments. ***P < 0.001; n.s, not significant; Student’s t-test. Scale bar, 20 μm