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Fig. S1. Perturbations of cell polarity in the Eya1-/- distal epithelium. (A,B,E,F) Immunohistochemistry of serial sections with specific antibodies shows loss of polarized apical localization of myosin IIB and F-actin at branching tips of E14 Eya1−/− lungs (arrowheads) compared with proximal epithelium (B,F, arrows) and control lungs (A,E). (C,D,G,H) Antibody staining for Par3/Par6 shows that they specifically localize to apical cell sides of wild-type distal epithelium (C,G; arrowheads), but localize to both basal (D,H; arrowheads) and apical cell sides in Eya1-/- distal epithelium. Broken lines represent the collagen IV-stained basement membranes. (I,J,N,O) Antibody staining of MLE15 cells shows severe reduction of polarized cortical localization of myosin IIB and F-actin (J,O; arrowheads), but increased disorganized actin stress fibers (O; arrows) after Eya1 knockout compared with control cells. (K,L,P,Q) Rescue of endogenous Eya1 function by co-transfection of murine siRNA and murine wild-type or enzymatically inactive mutant Eya1 constructs (not targeted by the siRNAs) for 48 hours in MLE15 cells reveals that loss of polarized cortical myosin IIB (K,L) and actin (P,Q) is dependent on Eya1 phosphatase activity. Arrows in Q show increased disorganized stress fibers. (M) Western blot for the experiments shown in I-L. Bars represent quantified western blot signals (mean±s.e.m., n=3, **P<0.001). Scale bars: 50 µm.
Fig. S2. Eya1 knockdown causes mislocalization of spindle-regulatory proteins in MLE-15 cells in vitro. (A,B) Immunofluorescence staining of mitotic MLE-15 cells for LGN (A) and pericentrin (B) shows polarized distribution and spindle-pole localization of LGN during mitosis. One of the spindle poles, which are seen by pericentrin-stained centrosomes (arrowheads), is positioned directly below LGN, indicative of a perpendicular alignment of the spindle (compare arrowheads in B with A). (C,D) LGN has a diffuse distribution in mitotic MLE-15 cells upon Eya1 knockdown (arrowheads). (E-H) 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 loss of polarized LGN localization is dependent on Eya1 phosphatase activity. There is non-polarized/diffuse LGN localization at different mitotic phases in F-H (arrowheads). (I,M) Quantification of mitotic MLE15 cells with polarized (I) or diffused (M) localization of LGN, NuMA or mInsc after different treatments for the experiments shown in A-H,J-P. This quantification is expressed as a percentage of all counted MLE15 cells. *Bars carrying the same letter (a,b,c) in I or M are significantly different from the control of the same protein (*P<0.05; ANOVA-Dunnett test). (J-P) Immunofluorescence staining shows polarized distribution/spindle-pole localization of Insc, NuMA and Par3 in mitotic MLE15 cells (J-L; arrowheads). (N-P) Insc, NuMA and Par3 have a diffuse/non-polarized distribution upon knocking down Eya1 in vitro (arrowheads). (Q-U) No apparent change in Eya1 expression after knocking down Numb, LGN, Insc or aPKCζ in vitro. Scale bars: 50 µm.
Fig. S3. Partial rescue of epithelial cell defects after genetic activation of Notch signaling in Eya1−/− lungs. (A,B,D,E,G,H) Immunohistochemistry shows reduced N-myc expression (E14.5, B; arrowheads), but increased differentiation markers SP-B and SP-C (E18.5, E,H; arrowheads) in Eya1-/- distal epithelium compared to control lungs (A,D,G; arrowheads). (C,F,I) In NICD; Spc-rtTA+/−-tet(O) Cre+/−Eya1-/- compound mutant lungs, the expression of N-myc (C; arrowheads), SP-B (F; arrowheads) and SP-C (I; arrowheads) is apparently restored into the control lung level (A,D,G; arrowheads). Scale bars: 50 µm.
