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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. 3. Gnas loss upregulates SHH by activating Yap activity.

Fig. 3.

(A) Quantified gene expression in Gnasf/f SMPs infected by indicated adenovirus. N=3 biological replicates. Western blotting analyses (right panel) of indicated proteins in Gnasf/f SMPs infected by indicated adenovirus. (mean±SD). **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (B) Representative μCT images of indicated tibia bones 6 weeks or 3 months after Ad-Cre injection. Red arrows indicate ectopic bone. White lines indicate section position. Lower panel: quantification of ectopic bone volume. N=5 biological replicates. Scale bar, 1 mm. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (C) Representative immunofluorescent images of OSX and tdTMT in ectopic bone sections of indicated mice, 6 weeks after Ad-Cre injection. Dashed lines indicate the interface between epidermis and dermis. Arrows: OSX+; tdTMT− cells. S: Skin; M: Muscle. Scale bar: 100 μm. (D) Top: HO induction and treatment schedules. Representative μCT images of tibia bones from indicated mice treated with the Yap inhibitor verteporfin (VP) five days per week starting from the day of Ad-Cre injection. Upper panel: Topical application of VP (0.2 mg/ml) around the injection site. Lower panel: intraperitoneal VP injection (2.5 mg/kg). Red arrows indicate ectopic bone. Bottom panel: quantification of ectopic bone volume. N=5 biological replicates. Scale bar: 1 mm. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (E) Representative μCT images of the indicated tibia bones. Doxycycline water treatment started right after Ad-Cre injection. Red arrows indicate ectopic bone. Right panel: quantification of ectopic bone volume. N=5 biological replicates. Scale bar: 100 μm. (F) qRT-PCR analysis of gene expression in the ectopic bone region from mice in (E). (mean±SD; N=3 biological replicates). NS: not significant. *p<0.05 **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. Ad-Cre injection was always performed in 4-week-old mice.