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. 2020 Mar 20;6(12):eaaz0368. doi: 10.1126/sciadv.aaz0368

Fig. 4. Gpr126 regulates osteoblast differentiation and function through the cAMP-CREB signaling pathway.

Fig. 4

(A) Intracellular cAMP level was decreased in Gpr126 deletion osteoblasts. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblasts. The cells were harvested and subjected to the cAMP enzyme-linked immunosorbent assay (ELISA) assay at day 7 of differentiation. **P < 0.01. n = 3. (B) Western blot analysis of the p-CREB, CREB, and OCN levels in BMSCs from indicated mice after 14 days of osteoblast differentiation. (C) Intracellular cAMP level was restored in Gpr126 deletion osteoblasts when treated with FSK. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblasts. The cells were treated with 10 μM FSK or vehicle control for 14 days, harvested, and subjected to the cAMP ELISA assay at day 14 of differentiation. **P < 0.01, ***P < 0.001. n = 3. (D) ALP enzyme activity (N = 5), ALP mRNA expression (n = 2), and OCN mRNA expression (n = 2) were restored in Gpr126 deletion osteoblasts treated with 1 μM FSK. The cells were harvested and subjected to ALP enzyme activity assay (at day 14) or real-time PCR assay (ALP at day 7 and OCN at day14). **P < 0.01, ***P < 0.001. (E) Osteoblast differentiation and mineralization were rescued in Gpr126 deletion osteoblasts when treated with FSK. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and control (Ctrl) littermates and differentiated into osteoblasts. ALP staining, von Kossa staining, and Alizarin red staining of BMSCs were performed after 7, 14, and 21 days of differentiation, respectively, while treated with or without 10 μM FSK. Scale bars, 5 mm. (F) ALP enzyme activity (N = 5), Alp mRNA expression (n = 2), and Ocn mRNA expression (n = 2) were restored in Gpr126 deletion osteoblasts treated with PTH(1–34). The cells were harvested and subjected to ALP enzyme activity assay (at day 14) or real-time PCR assay (Alp at day 7 and Ocn at day14). **P < 0.01, ***P < 0.001. (G) Osteoblast differentiation and mineralization were rescued in Gpr126 deletion osteoblasts when treated with PTH(1–34). BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblast. ALP staining, von Kossa staining, and Alizarin red staining of BMSCs were performed after 7, 14, and 21 days of differentiation, respectively, while treated with or without PTH(1–34) (80 μg/kg). Scale bars, 5 mm.