Fig. 6.

Inhibition of Notch signaling in Eya1^−/− lung distal epithelium. (A-F) Immunohistochemistry with specific antibodies shows reduced staining of activated-Notch1 (B), Hes1 (D) and Hes5 (F) in E14 Eya1^−/− distal epithelium (arrowheads) compared with control lungs (A,C,E; arrowheads). (G) Western blots show reduction of activated-Notch1 and Hes-5 in E14 Eya1^−/− lungs. (H) Western blot of activated-Notch1 for experiments shown in I-M. Blue numbers in G,H,S represent relative band intensity. (I,J) Immunocytochemistry shows reduced activated-Notch1 expression in MLE-15 after Eya1 knockdown. (K,L) Rescue of endogenous Eya1 function by co-transfection of murine siRNA and murine wild-type or enzymatically inactive mutant Eya1 constructs for 48 hours in MLE15 cells reveals that Notch1 signaling/activity is dependent on Eya1 phosphatase activity. (M) Inhibition of aPKCζ in Eya1 siRNA-transfected MLE15 cells rescues activated-Notch1 expression. (N) Mean fluorescence intensity of Hes1 staining for experiments showing in O-R. *Significantly different from control (P<0.05; ANOVA-Dunnett test). Error bars indicate s.e.m. (O-Q) Immunocytochemistry of MLE-15 cells shows decreased Hes1 expression after Eya1 knockdown (O,P), but increased Hes-1 expression upon Eya1 overexpression (Q; arrowheads). (R) Hes1-positive cells with strong nuclear staining further increase after co-transfection of Numb siRNA and wild-type Eya1 expression vector in MLE-15 cells (arrowheads). (S) Western blot of Hes1 for experiments showing in O-R. (T) Quantitation of Hes1-positive cells, which is expressed as a percentage of all counted MLE15 cells, of the experiments shown in O-R. *Significantly different from control (P<0.05; ANOVA-Dunnett test). Data are mean±s.e.m. Scale bars: 50 μm.