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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of <t>Wnt3,</t> β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.
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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of <t>Wnt3,</t> β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.
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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of <t>Wnt3,</t> β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.
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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of <t>Wnt3,</t> β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.
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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of <t>Wnt3,</t> β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.
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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of <t>Wnt3,</t> β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.
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A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of Wnt3, β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Communications Biology

Article Title: Exercise increases bone mass by lactate/Gpr81 signaling pathway

doi: 10.1038/s42003-025-08957-1

Figure Lengend Snippet: A The BMM of mice from the vehicle and NG-25-treated groups were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, followed by Trap staining. Scale bars, 200 μm. B Quantifying osteoclast number and Trap-positive area in ( B ). n = 6. *** p < 0.001. Two-way ANOVA was performed followed by Dunnett’s test for multiple comparisons with experiments repeated independently three times. C RT-qPCR analysis of Trap , Nfatc1 and CTSK mRNA levels, * p < 0.05, ** p < 0.01, *** p < 0.001. n = 4. D BMM of mice from the vehicle and NG-25-treated cells were taken to induce osteoclast differentiation for 6 days with or without 10 mM lactate intervention, and Western blots analysis of p65, p-p65, TAK1 and Gpr81 proteins levels in osteoclasts. E Quantification of TAK1, Gpr81 and p-p65 protein expression levels. * p < 0.05, ** p < 0.01. n = 6. F Lactate promotes osteogenic differentiation and mineralization, and BMSC differentiation and mineralization are inhibited in iCRT(Wnt inhibitor) treated mice. The BMSC were differentiated for 7, 14 and 21 days, respectively, and then stained for ALP, von Kossa’s and Alizarine Red in the presence or absence of 10 mM lactate. G BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactate intervention, respectively, and RT-qPCR analysis of Runx2 , ALP , and Osterix mRNA levels in osteoblasts. All data are presented as mean ± SD. n = 6. ** p < 0.01., *** p < 0.001. H BMSC of mice from the vehicle and iCRT3-treated groups were taken to induce osteogenic differentiation for 14 days with or without 10 mM lactic acid intervention, and Western blots analysis of Wnt3, β-catenin, and OCN proteins levels in osteoblasts. I Quantification of Wnt3, β-catenin and OCN protein expression levels. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked with BSA and incubated with specific antibodies, including an osteocalcin antibody (Bioss, bs-0470R, 1:1000) and Wnt3 antibody (Proteintech, 28156-1-A, 1:1000).

Techniques: Staining, Quantitative RT-PCR, Western Blot, Expressing