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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. 2. Gnas loss induces bone formation non-cell autonomously by inducing SHH expression and secretion.

Fig. 2.

(A) Representative images of ALP and von Kossa staining of wild-type SMPs, cultured with conditioned medium (CM) from indicated adenovirus-infected SMPs for 7 days and 21 days, respectively. N=3 biological replicates. Two sets were shown. (B) Left panel: quantification of ALP activity from (a). Right panel: qRT-PCR analysis of gene expression in WT SMPs cultured with indicated CM for 7 days (mean±SD; N=3 biological replicates). **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (C) qRT-PCR analysis of Hh gene expression in Gnasf/f SMPs, 4 days after adenovirus infection. SHH protein, phosphorylated Creb (pCreb), and total Creb protein, were detected by Western blotting (right panel). β-ACTIN: loading control. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. NS: not significant. (D) Secreted SHH protein detected by Western blotting of CM from two independent sets of Gnasf/f SMPs, 4 days following adenovirus infection. (E) Left panel: ALP staining of wild-type SMPs, cultured with indicated CM and SHH monoclonal blocker (5E1; 200 ng/ml) for 7 days. Middle panel: quantification of ALP activiy. Right panel: qRT-PCR analysis of gene expression. N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (F) Left panel: ALP staining of the indicated SMPs 7 days after adenovirus infection. Right panel: quantification of ALP activity. N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (G) Representative μCT images of the indicated tibia bone 6 weeks or 3 months after subcutaneous Ad-Cre injection at 4 weeks of age. Red arrows indicate ectopic bone. White lines indicate section position. Lower panel: quantification of ectopic bone volume. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. N=5 biological replicates. Scale bar, 1 mm. (H) Representative immunofluorescent images of OSX and tdTMT expression in ectopic bone sections of indicated genotypes, 3 months after Ad-Cre injection of 4-week-old mice. Arrows: OSX+; tdTMT− cells. S: Skin; M: Muscle. Dashed lines indicate the interface between epidermis and dermis. N=5 biological replicates. Scale bar: 100 μm.