Fig. 4

PKA activation and SNAP23 are both required for EDA-mediated MG growth. A Immunohistochemical (IHC) staining shows the expression of K14 (green) and other indicated proteins (red) in WT or EDA^−/− eyelids at embryonic day 17.5 (E17.5). Arrows indicate MG germs. Scale bar, 10 μm. B A graphic drawing shows the workflow of eyelid organotypic culture followed by indicated treatment and immunoblotting / IHC study. C Immunoblotting shows the intensity of PKA activity with indicated treatment of EDA, H89, or AAV-mediated SNAP23 KD (shSNAP23) in EDA^−/− eyelid cultures. D Immunoblotting shows the levels of indicated proteins in PM fraction from identical tissue cultures and treatment as in C. E–F IHC shows the expression of K14 (green) and LEF1 (red) after 2 days culture of WT eyelids isolated at E15.5. MG germs indicated by dotted lines. The MG length were calculated as described (see Methods) and quantified as shown in F. G–H As in E and F, except with transfection of AAV-scramble (Scr) or AAV-shSNAP23. Error bars indicate mean ± SD from at least 20 MGs in total 3 independent experiments. ***P < 0.001; Student’s t-test. Scale bar, 20 μm