Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) Representative flow cytometry plots showing the gating strategy for isolating MuSCs (TER119 – CD45 – CD31 – Sca-1 – CD106 + ) from muscles of WT mice at 1 dpi. ( B ) Representative immunofluorescence images showing MyHC + myotubes (green) and nuclei (Hoechst; blue) in myoblast cultures with or without 100 ng/mL activin A treatment on day 7. MuSCs were expanded for 4 days to over 90% confluency and differentiated for 3 days. The boxed areas are shown at higher magnification (×4) in the adjacent lower panels. Scale bars: 100 μm. ( C and D ) Quantification of myotube fusion in B , showing the number of nuclei per myotube ( C ) and the percentage of myotubes with more than 4 nuclei ( D ). ( E ) Relative count of myoblasts on the indicated days. MuSCs were isolated on day 0 and then cultured for 1 to 3 days with or without 100 ng/mL activin A. ( F ) RT-qPCR analysis showing relative Myog mRNA expression in myoblasts treated with or without 100 ng/mL activin A for 2 days under differentiation conditions. The P values were calculated using unpaired 2-tailed t test ( C , D , and F ) or 2-way ANOVA with Bonferroni correction for multiple comparisons ( E ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM.

Article Snippet: To analyze activin A expression in muscle tissue, sections were stained with APC anti-F4/80 antibody (clone BM8; BioLegend), anti-mouse activin A antibody (catalog AF338; R&D Systems), and corresponding Alexa Fluor 488 secondary antibody (catalog A-11029; Thermo Fisher Scientific).

Techniques: Flow Cytometry, Muscles, Immunofluorescence, Isolation, Cell Culture, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) UMAP visualization of cells isolated from muscle at 1 dpi. Phylogenetic tree showing cluster hierarchy and cell numbers (in parentheses). ( B ) Heatmap showing the top 10 DEGs by log 2 FC (adjusted P < 0.001) across clusters. Typical cell surface molecules were selected for naming. Expression of Ly6C is shown in . ( C ) UMAP showing the expression of Inhba in each cluster. ( D ) Dot plot showing the relative Inhba expression across 3 subclusters of MDMs. ( E and F ) RT-qPCR results showing relative Inhba mRNA expression in muscles at 1 dpi from mice treated with 150 μL vehicle ( n = 7) or clodronate ( n = 9) ( E ) and from mice treated with 0.25 mg isotype IgG ( n = 8) or 0.25 mg anti-Ly6G antibodies ( n = 7) ( F ). ( G ) Gating strategy for sorting 4 subpopulations of Ly6C hi CX3CR1 lo MDMs based on the expression of PDPN and CD9. ( H ) Flow cytometric analysis confirming the purity of sorted cells. ( I ) RT-qPCR results showing relative Inhba expression in indicated subpopulations sorted from muscles on 1 dpi ( n = 4 for each). The expression in the PDPN – CD9 – fraction was set to 1. ( J ) ELISA results showing the concentration of activin A in the culture supernatants of indicated MDMs sorted from muscles on 1 dpi. Supernatants were collected 48 hours after seeding. ( K ) Flow cytometric histogram showing IL-7R expression in indicated MDMs at 1 dpi. The P values were calculated using unpaired 2-tailed t test ( D , E , and J ) or 1-way ANOVA with Tukey’s multiple-comparison test ( I ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice ( D , E , I , and J ).

Article Snippet: To analyze activin A expression in muscle tissue, sections were stained with APC anti-F4/80 antibody (clone BM8; BioLegend), anti-mouse activin A antibody (catalog AF338; R&D Systems), and corresponding Alexa Fluor 488 secondary antibody (catalog A-11029; Thermo Fisher Scientific).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Muscles, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) Representative microCT images of the hind limbs of uninjured ( n = 3) and injured gHO mice treated with vehicle ( n = 4) or ACVR1 kinase activity inhibitor ( n = 5) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( B and C ) Quantification showing HO volume ( B ) and bone mineral content ( C ) in the gHO mice treated with vehicle or ACVR1 inhibitor. ( D ) Experimental timeline for the coculture of FAPs with Mrep. FAPs, non-FAPs, Mrep, and other cells (the remaining CD45 + cells) were sorted from muscles of gHO mice at 1 dpi. Anti–activin A antibody (1 mg/mL) was used to neutralize activin A. ( E ) Alizarin red S staining of the coculture experiment in D . Representative data from 3 independent experiments are shown. ( F and G ) Representative microCT images ( F ) and quantification ( G ) of HO in Acvr1 Q207D -induced HO of Inhba fl/fl mice ( n = 26) and Inhba fl/fl LysM-Cre mice ( n = 17) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( H ) RT-qPCR results showing the relative Inhba expression in the muscles at 1 dpi from the mice treated with DMSO ( n = 5) or TAK-242 ( n = 5). ( I – K ) Representative microCT images ( I and J ) and quantification ( K ) of HO in gHO mice treated with vehicle ( n = 10), TAK-242 ( n = 7), and clodronate ( n = 7) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. The P values were calculated using unpaired 2-tailed t test ( B , C , G , and H ) and 1-way ANOVA with Tukey’s multiple-comparison test ( K ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice.

Article Snippet: To analyze activin A expression in muscle tissue, sections were stained with APC anti-F4/80 antibody (clone BM8; BioLegend), anti-mouse activin A antibody (catalog AF338; R&D Systems), and corresponding Alexa Fluor 488 secondary antibody (catalog A-11029; Thermo Fisher Scientific).

Techniques: Activity Assay, Muscles, Staining, Quantitative RT-PCR, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) Representative microCT images of the hind limbs of uninjured ( n = 3) and injured gHO mice treated with vehicle ( n = 4) or ACVR1 kinase activity inhibitor ( n = 5) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( B and C ) Quantification showing HO volume ( B ) and bone mineral content ( C ) in the gHO mice treated with vehicle or ACVR1 inhibitor. ( D ) Experimental timeline for the coculture of FAPs with Mrep. FAPs, non-FAPs, Mrep, and other cells (the remaining CD45 + cells) were sorted from muscles of gHO mice at 1 dpi. Anti–activin A antibody (1 mg/mL) was used to neutralize activin A. ( E ) Alizarin red S staining of the coculture experiment in D . Representative data from 3 independent experiments are shown. ( F and G ) Representative microCT images ( F ) and quantification ( G ) of HO in Acvr1 Q207D -induced HO of Inhba fl/fl mice ( n = 26) and Inhba fl/fl LysM-Cre mice ( n = 17) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( H ) RT-qPCR results showing the relative Inhba expression in the muscles at 1 dpi from the mice treated with DMSO ( n = 5) or TAK-242 ( n = 5). ( I – K ) Representative microCT images ( I and J ) and quantification ( K ) of HO in gHO mice treated with vehicle ( n = 10), TAK-242 ( n = 7), and clodronate ( n = 7) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. The P values were calculated using unpaired 2-tailed t test ( B , C , G , and H ) and 1-way ANOVA with Tukey’s multiple-comparison test ( K ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice.

Article Snippet: For the inhibition of activin A activity, 1 mg/mL anti–activin A antibody (catalog AF338; R&D Systems) was added to the osteogenic differentiation medium.

Techniques: Activity Assay, Muscles, Staining, Quantitative RT-PCR, Expressing, Comparison