Fig. 4: CLCF1 inhibits osteoclast differentiation and stimulates osteoblast differentiation.
From: Exercise-induced CLCF1 attenuates age-related muscle and bone decline in mice
A–C BMMs were treated with different concentrations of CLCF1 in the presence of M-CSF and RANKL for 3 days. A The numbers of TRAP-positive osteoclasts were counted (n = 4; biological replicates, p = 3.32e-5, 4.6e-9, 2.0e-10). Scale bar, 100 μm. B Real-time PCR analyses for the expression of the indicated genes (n = 3; p = 2.2e-7, 8.0e-4, 0.4408, 1.0e-9, 1.0e-9). C Western blot for the expression of the indicated molecules. D TRAP staining and quantification of osteoclasts differentiated from BMMs transduced with pMX-IRES-EGFP or CLCF1 and CRLF1 retrovirus for 3 days (n = 3; biological replicates, p = 0.0081). Scale bar, 100 μm. E Schemes of RANKL-induced bone loss (created in BioRender.com). F Representative µCT images of the trabecular and cortical compartments of distal femoral metaphyseal regions. Scale bar, 1 mm. G–J Measurement of trabecular bone volume /total volume (BV/TV, p = 5.0e-5, 0.0052, 0.019), trabecular thickness (TbTh, p = 0.0174, 0.0032), trabecular separation (TbSp, p = 0.0329, 0.0007), trabecular number (TbN, p = 0.0014, 0.000046) (n = 4). K–M Osteoblast precursors differentiated with OGM for 3 or 6 days in the presence or absence of CLCF1 (100 ng/mL). K ALP assay after 3 days (n = 3; biological replicates, p = 0.0130). L Alizarin red staining and quantification at 6 days (n = 3; p = 0.0208). M PCR analyses for the expression of the indicated genes (n = 3; p = 1.0e-4, 1.0e-10). N, O Osteoblasts transduced with pMX-IRES-EGFP or CLCF1 and CRLF1 retrovirus were differentiated with OGM for 3 or 6 days. N ALP activity was measured (n = 3; p = 0.0198). O Alizarin red staining and quantification (n = 3; p = 0.0064). P Schemes of BMP2-induced ectopic bone formation (created in BioRender.com). Q Representative µCT images of the ectopic bones. Scale bar, 1 mm. R Measurement of BV and BV/TV (n = 6; p = 0.0451, 0.0437). Data are presented as means ± s.e.m.; two-tailed unpaired Student’s t-test (A, D, R); one-way ANOVA with post hoc Tukey’s multiple comparison test (G–J); or two-way ANOVA with post hoc Tukey’s multiple comparison test (B, K–O). *p < 0.05, **p < 0.005 and ***p < 0.001. Source data are provided as a Source Data file.
