Fig 1: CTX-induced muscle injury generated an osteoinductive environment. (A) Phosphorylated Smad1 level in muscle tissue lysates measured by pSMAD1 ELISA at different time points after CTX injection demonstrated the activation of BMP signalling (**, p < 0.01; n = 3). (B) PCR analysis showed the upregulation of osteogenic marker gene expression in muscle tissue after CTX injection with the expression of different genes peaking at different time points. (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; n = 3). Data are normalized to uninjected Day 0 control mice. (C) Western blot analysis of phosphorylated Smad1/5 protein and total Smad1 levels revealed the time course of BMP signalling activation in MDSCs cultured in GM and muscle tissue–derived CM. (D) PCR analysis of osteogenic marker gene and BMP type I receptor gene expression in MDSCs. Significantly higher OSX, ALP and BMPR1B expression was detected in MDSCs cultured in CTX CM compared with CTL CM (*, p < 0.05; n = 3). Data are normalized to MDSCs cultured in GM.
Fig 2: Inhibition of BMP-7 activity suppressed osteogenesis in vitro and reduced HO formation in vivo. (A) Osteogenesis of MDSCs in vitro. ALP activity measured in different groups of cell lysates showed that treatment with BMP-7–neutralizing antibody suppressed the osteogenesis-promoting effect of CTX CM (*, p < 0.05; **, p < 0.01; ****, p < 0.0001; n = 3). Iso: isotype control antibody treatment group; Ab: BMP-7–neutralizing antibody treatment group. (B) BMP signalling in vitro. Phosphorylated Smad1 level in cell lysates measured by pSMAD1 ELISA showed that activation of BMP signalling was inhibited with addition of BMP-7–neutralizing antibody (****, p < 0.0001; n = 3). (C) HO formation in vivo. microCT imaging and analysis showed significant reduction in HO volume upon BMP-7–neutralizing antibody treatment compared with isotype control antibody treatment (*, p < 0.05; n = 4). No significant difference in bone mineral density was found between BMP-7–neutralizing antibody and isotype control antibody treatment groups.
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