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. Author manuscript; available in PMC: 2022 Jun 23.
Published in final edited form as: Sci Transl Med. 2021 Jun 23;13(599):eabb2233. doi: 10.1126/scitranslmed.abb2233

Fig. 4. Yap and SHH form a positive feedback loop.

Fig. 4.

(A) ALP staining of Gnasf/f SMPs, 7 days after indicated Ad-GFP or Ad-Cre infection and treatment with VP (200 ng/ml) or a SHH monoclonal blocker (200 ng/ml). Right panel: quantification of ALP activity. N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (B) SHH protein detected by Western blotting of Gnasf/f SMPs, treated as in (a). GAPDH: loading control. (C) ALP staining of WT SMPs cultured for 7 days in indicated conditioned medium. Right panel: quantification of ALP activity. N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (D) Western blotting analysis of secreted SHH protein in the indicated CM. (E Representative immunofluorescent images of ectopic bone sections of the indicated genotypes, 3 months (left panel) or 8 months (right panel) after Ad-Cre injection. Dashed lines indicate the interface between epidermis and dermis in (E-G). Arrows: Yap+; tdTMT − or SHH+; tdTMT− cells. S: Skin; M: Muscle. Scale bar: 100 μm. (F, G) Representative immunofluorescent images of Yap (F), SHH (G), and tdTMT in the ectopic bone section from the indicated mice 6 weeks after Ad-Cre injection. Arrows: Yap+; tdTMT− or SHH+; tdTMT− cells. S: Skin; M: Muscle. Scale bar: 100 μm. Ad-Cre injection was always performed in 4-week-old mice.