Fig. 6. Numb loss blocks the activation of Gli transcription activator.

a Immunofluorescence staining of endogenous Gli2 in the cilia of WT or Numb KO cells. The cells were treated in low serum medium for 24 h, with or without Shh. Enlarged views of highlighted areas are displayed at the bottom. Arrows point to Gli2 fluorescence signal at the cilium tips. b Quantification of Gli2 fluorescence intensity at the cilium tips. A total of 50 cilia were quantified per condition. RFU, relative fluorescence unit. c, d Western blot results and quantification of Gli3 processing in WT and Numb KO cells. Cells were stimulated with Shh for 24 h prior to harvest. e Schematic of Numb’s role during the activation of Hh signaling. Upon Shh binding to Ptch1, Numb in the ciliary pocket (blue lines) recruits Ptch1-Shh complex into clathrin-coated vesicles, thereby facilitating the removal of Ptch1 from the cilium. Ptch1’s efficient clearance from the cilium sets the stage for the complete activation of Smo and Gli2 transcription factors. This activation ultimately culminates in the maximal activation of Hh signaling. In the absence of Numb, Ptch1 remains in the cilium even after it binds to Shh. This leads to only a partial activation of Smo, which ceases Gli3R production but is insufficient to activate Gli2. As a result, Hh signaling is only moderately activated. Results from three independent experiments are analyzed. Data are shown as mean ± SD. Statistics in (b, d): Two-way ANOVA with multiple comparisons (Tukey test). ns, not significant. Source data are provided as a Source Data file.