Fig. 3.

Overexpressed Disp^tg locates to the cell surface and restores Shh release from Disp^−/− cells. (A,A′) Representative confocal planes of Disp^−/− cells expressing Shh (A, red) or Disp^tg (A′, red). Both transgenes were secreted to the cell surface. Nuclei were counterstained with DAPI (blue). Dashed lines indicate the border of cytoplasmic eGFP signals (green). Images are representative of three experiments. Scale bars: 2 µm. (B) Co-expressed transgenic Disp^tg enhanced processed Shh release from Disp^−/− cells (c) into the medium (m). Transgenic Disp^ΔL2, which lacks most of the second extracellular loop, did not release significantly increased amounts of truncated Shh. Empty-vector (EV)-transfected Disp^−/− cells served as negative controls. (B′) Quantified relative processed Shh release, as shown in B. Data are mean±s.d. n=10. **P<0.01; ns, not significant; P=0.377 for Disp^ΔL2 (one-way ANOVA with Dunnett's multiple comparison post hoc test). (C) Co-expressed transgenic Disp^tg or Disp^ΔL2 did not significantly increase Shh release from nt Ctrl cells. (C′) Quantified relative processed Shh release as shown in C. Data are mean±s.d. n=14. ns, not significant (one-way ANOVA with Dunnett's multiple comparison post hoc test). In B and C, arrows indicate solubilized truncated Shh from Disp^tg- or Disp^ΔL2-expressing cells and the arrowhead indicates solubilized Shh from EV-transfected cells. Anti-β-actin blots (αβ-actin) and Ponceau S staining of residual serum albumin (PonS) serve as loading controls.