Gli1⁺ Cells Residing in Bone Sutures Respond to Mechanical Force via IP₃R to Mediate Osteogenesis

Figure 5

Gli1⁺ cells participate in mechanical force-mediated JBMMSC osteogenesis through an IP₃R-induced intracellular calcium concentration increase. (a) Experimental procedure: Gli1-mT/mG mice were treated with tamoxifen for 4 consecutive days and were sacrificed after 7 days to isolate JBMMSCs. After 6 days of incubation, cells were treated with GANT61 (stretch+GANT61) or vehicle (stretch). (b) Flow cytometry analysis of the proportion of Gli1⁺ cells in JBMMSCs showed a decrease of Gli1⁺ cells in the GANT61-treatment; . (c) The proportion of Gli1⁺ cells was lower in the stretch+GANT61 group; ; . (d) ALP activity of JBMMSCs treated with GANT61 was reduced after osteogenic induction for 7 days. Mineralized nodules formed by JBMMSCs were detected using alizarin red staining after osteogenic induction for 14 days. (e) The abundance of mineralized nodules in the stretch+GANT61 group was lower; ; . (f) Western blot analysis showed downregulation of ALP, RUNX2, Col1, and IP₃R protein expression in JBMMSCs treated with GANT61 and mechanically stretched, compared with cells subjected only to mechanical stretching; . (g) RT-qPCR showed downregulation of ALP, RUNX2, and Col1 RNA expression in JBMMSCs treated with GANT61 and mechanically stretched, compared with cells subjected only to mechanical stretching. ; ; . (h) RT-qPCR showed downregulation of IP₃R1, IP₃R2, and IP₃R3 RNA expression in JBMMSCs treated with mechanical stretching and GANT61, compared with cells subjected only to mechanical stretching; ; ^ns; . (i) Intracellular calcium concentration visualized at mean fluorescence intensity was decreased in the stretch+GANT61 group. ; .

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