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: Nature medicine

Article Title: An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

doi: 10.1038/nm.3468

Figure Lengend Snippet: RAP-011 treatment reduces ineffective erythropoiesis in thalassemic mice. (a–c) Spleen weight (a), total spleen cell number (b) and bone marrow cellularity (c) of thalassemic mice treated for 5, 10, 30 or 60 d with RAP-011 or PBS. (d) Bone marrow erythroblast number and distribution observed in H&E-stained cross-sections of bones of RAP-011–treated or PBS-treated thalassemic mice (60 d of treatment). (e) Bone marrow and spleen erythroblast number in RAP-011– or PBS-treated mice (30 d of treatment). (f) Erythroblast differentiation in bone marrow and spleen harvested 5–30 d after treatment with RAP-011 and evaluated by CD71 and Ter-119 staining and forward scatter (FSC) distribution. The percentage of different erythroblast populations is shown. Box-and-whisker plots show means and maximal and minimal values. (g) Representative flow cytometry analysis of spleen and bone marrow erythroblast subset distribution in RAP-011– and PBS-treated thalassemic mice (30 d of treatment). FSC-A, forward scatter area. (h) An index of ineffective erythropoiesis established by calculating the ratio of Ery.B and Ery.C percentage populations. Box-and-whisker plots show means and maximal and minimal values. (i,j). Biochemical analysis of parameters of ineffective erythropoiesis in sera of thalassemic mice treated for up to 60 d with RAP-011 or PBS: direct bilirubin (i) and total bilirubin (j). All data are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005; n = 5 mice per group for one out of three independent experiments.

Article Snippet: For immunohistochemistry and confocal analysis, sections were processed for antigen retrieval as indicated by each manufacturer and incubated overnight with primary antibodies against GDF11 proform (ab71347, Abcam; 1/100 dilution), mature GDF11 (sc28910, Santa Cruz; 1/20 dilution), activin A (AF338, R&D Systems; 1/100 dilution), activin B (MAB659, R&D Systems; 1/100 dilution), activin receptor II (sc-25451, Santa Cruz; 1/100 dilution), phospho-Smad1/5 (#9511, Cell Signaling; 1/100 dilution), phospho-Smad2 (#3101, Cell Signaling; 1/100 dilution), Ter-119 (#553673, BD Biosciences; 1/100 dilution), CD71 (136800, Life Technologies; 1/200 dilution) or F4/80 (sc-377009, Santa Cruz Biotechnology; 1/50 dilution).

Techniques: Staining, Whisker Assay, Flow Cytometry

Journal: Nature medicine

Article Title: An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

doi: 10.1038/nm.3468

Figure Lengend Snippet: GDF11 inactivation promotes terminal erythropoiesis. (a) Immunohistochemical staining of phosphorylated Smad2/3 (p-Smad2/3), p-Smad1/5 and ActRIIA and ActRIIB in spleen samples from wild-type and thalassemic mice treated with PBS or RAP-011 for 30 d. (b,c) Confocal micrographs showing sections of spleen red pulp from wild-type and thalassemic mice. p-Smad2/3 protein expression (green), Ter-119+ erythroblasts (blue) and F4/80+ (b) and CD71+ (c) cells (red) are shown. (d) Confocal micrographs showing sections of spleen and bone marrow from thalassemic mice. p-Smad2/3 protein expression (green) and Ter-119+ erythroblasts (blue) are shown. (e–g) Erythroblast cultures, derived from cells from thalassemic mice, treated with pyrrolidine dithiocarbamate (PDTC) (5 μM), RAP-011 (10 μg/ml) or PBS as a vehicle for 48 h. (e) Flow cytometry analysis showing intracellular GDF11 levels after treatment with PDTC or RAP-011. (f) Erythroblast differentiation after treatment with PDTC. Cells were classified as immature (Ter-119+CD71+) or mature (Ter-119+CD71−) erythroblasts. (g) ROS levels in bone marrow–derived thalassemic erythroblasts after treatment with RAP-011 or PDTC. (h) ROS production after treatment of erythroblast cultures from bone marrow of thalassemic mice with rGDF11, rGDF15 or rGDF8 (5 or 50 ng/ml). ROS generation was measured by FACS using DCFH. (i) Erythroblast cultures, derived from bone marrow of thalassemic mice, treated with neutralizing antibodies against activin A, activin B or GDF11 propeptide. Flow cytometry analysis of erythroblast differentiation using CD71 and Ter-119 staining and FSC distribution is shown. Cells were classified as ProE (Ter-119dimCD71+), immature (Ter-119+CD71+) and mature (Ter-119+CD71−) erythroblasts. (j) Erythroblast differentiation in samples treated with rGDF11 (100 ng/ml) for 48 h, as evaluated by CD71 and Ter-119 staining and FSC distribution. The percentages of immature (Ter-119+CD71+) and mature (Ter-119+CD71−) erythroblast populations are shown. All data are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01; n = 3–5 mice per group for one out of three independent experiments.

Article Snippet: For immunohistochemistry and confocal analysis, sections were processed for antigen retrieval as indicated by each manufacturer and incubated overnight with primary antibodies against GDF11 proform (ab71347, Abcam; 1/100 dilution), mature GDF11 (sc28910, Santa Cruz; 1/20 dilution), activin A (AF338, R&D Systems; 1/100 dilution), activin B (MAB659, R&D Systems; 1/100 dilution), activin receptor II (sc-25451, Santa Cruz; 1/100 dilution), phospho-Smad1/5 (#9511, Cell Signaling; 1/100 dilution), phospho-Smad2 (#3101, Cell Signaling; 1/100 dilution), Ter-119 (#553673, BD Biosciences; 1/100 dilution), CD71 (136800, Life Technologies; 1/200 dilution) or F4/80 (sc-377009, Santa Cruz Biotechnology; 1/50 dilution).

Techniques: Immunohistochemical staining, Staining, Expressing, Derivative Assay, Flow Cytometry

Journal: Nature medicine

Article Title: An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

doi: 10.1038/nm.3468

Figure Lengend Snippet: GDF11 is overexpressed in β-thalassemia and is associated with ineffective erythropoiesis. (a) Gdf11 mRNA levels evaluated by qPCR in spleen and bone marrow erythroblasts from wild-type and thalassemic mice (n = 4 for each). AU, arbitrary units. (b) Representative activin A, activin B and GDF11 immunohistochemical staining of spleen sections from wild-type and thalassemic mice treated with PBS or RAP-011 for 30 d. (c) Representative activin A, activin B and GDF11 immunohistochemical staining of spleen sections from wild-type C57BL/6 mice under conditions of normoxia, hypoxia and hemolytic anemia (aRBC). (d) Gdf11, activin A (Inhba) and activin B (Inhbb) mRNA levels evaluated by qPCR from the spleen of wild-type C57BL/6 mice under conditions of normoxia, hypoxia and hemolytic anemia (n = 4 for each). (e,f) Confocal micrographs showing splenic sections from wild-type and thalassemic mice. GDF11 protein expression is shown in green, Ter-119+ erythroblasts in blue and F4/80+ (e) and CD71+ (f) cells in red. (g) Confocal micrographs showing sections from spleen and bone marrow of thalassemic mice. GDF11 protein expression is shown in green and nuclei (DAPI) in blue. Scale bars, 50 μm. (h) Detection of ActRIIA Fc–bound ligands in sera from healthy individuals (n = 8) and subjects with thalassemia (n = 16). (i) Detection of ActRIIa Fc–bound ligand GDF11 in sera from wild-type (n = 6) mice and thalassemic mice (n = 5). All data are expressed as the mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 for one out of three independent experiments.

Article Snippet: For immunohistochemistry and confocal analysis, sections were processed for antigen retrieval as indicated by each manufacturer and incubated overnight with primary antibodies against GDF11 proform (ab71347, Abcam; 1/100 dilution), mature GDF11 (sc28910, Santa Cruz; 1/20 dilution), activin A (AF338, R&D Systems; 1/100 dilution), activin B (MAB659, R&D Systems; 1/100 dilution), activin receptor II (sc-25451, Santa Cruz; 1/100 dilution), phospho-Smad1/5 (#9511, Cell Signaling; 1/100 dilution), phospho-Smad2 (#3101, Cell Signaling; 1/100 dilution), Ter-119 (#553673, BD Biosciences; 1/100 dilution), CD71 (136800, Life Technologies; 1/200 dilution) or F4/80 (sc-377009, Santa Cruz Biotechnology; 1/50 dilution).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Nature medicine

Article Title: An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia

doi: 10.1038/nm.3468

Figure Lengend Snippet: ActRIIA trap therapy promotes early-stage erythroblast apoptosis in thalassemic mice. (a) TUNEL staining of CD71+ erythroblasts. Confocal micrographs showing sections from wild-type and thalassemic mice (n = 3 mice for each group) treated with RAP-011 or PBS for 30 d. TUNEL+ staining is shown in green, Ter-119+ in blue and CD71+ in red. (b) Flow cytometric quantification of Fas+ and FasL+ erythroblast populations (Ery.A, Ery.B and Ery.C) from bone marrow and spleen of thalassemic mice treated with RAP-011 or with PBS for 30 d (n = 5 mice for each group). Representative FACS histograms of Fas and FasL staining in Ery.B spleen cells are also shown. (c) The percentage of Fas and FasL cells in cultured thalassemic erythroblast cells at the indicated stages of differentiation after treatment without or with neutralizing antibodies specific for GDF11 (GDF11 Ab), activin A (ActA Ab) or activin B (ActB Ab) (one out of three independent experiments, n = 3 mice for each group). (d) Quantification of apoptosis (annexin V (AV) and 7-AAD staining) in cultured erythroblasts from thalassemic mice treated with neutralizing antibodies against GDF11, activin A or activin B (one out of three independent experiments, n = 3 mice for each group). All data are expressed as the mean ± s.e.m. *P < 0.05 and **P < 0.01.

Article Snippet: For immunohistochemistry and confocal analysis, sections were processed for antigen retrieval as indicated by each manufacturer and incubated overnight with primary antibodies against GDF11 proform (ab71347, Abcam; 1/100 dilution), mature GDF11 (sc28910, Santa Cruz; 1/20 dilution), activin A (AF338, R&D Systems; 1/100 dilution), activin B (MAB659, R&D Systems; 1/100 dilution), activin receptor II (sc-25451, Santa Cruz; 1/100 dilution), phospho-Smad1/5 (#9511, Cell Signaling; 1/100 dilution), phospho-Smad2 (#3101, Cell Signaling; 1/100 dilution), Ter-119 (#553673, BD Biosciences; 1/100 dilution), CD71 (136800, Life Technologies; 1/200 dilution) or F4/80 (sc-377009, Santa Cruz Biotechnology; 1/50 dilution).

Techniques: TUNEL Assay, Staining, Cell Culture

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) Confocal imaging of ITGα2, Vimentin, and K8 in an IDC patient tissue section. Green contour: cluster of cancer cells with protrusive morphology; green arrowheads: basal-like cells (K8-low) at the tumor–stroma interface with high ITGα2 expression. White contour: cluster of cancer cells lacking basal-like cells (K8-high), with low ITGα2 expression; magenta arrowheads. White arrowheads: fibroblast-like cells (elongated, spindle-shaped). (B) Quantification of mean gray values for ITGα2 and Vimentin in basal-like (n = 33), luminal-like (n = 32), and fibroblast-like (n = 32) cells from one IDC patient tissue section. (C) Representative brightfield images of MMTV-PyMT organoids (ITGα2-WT or ITGα2-KO, gRNA1 and gRNA2) cultured in 3D Collagen I. Black arrowheads: invasive strands. (D) Percentage of organoids exhibiting one or more invasive strands in ITGα2-WT and ITGα2-KO (clones 1 and 2 from gRNA1) MMTV-PyMT organoids. (E) qPCR analysis of classical TGF-β and EMT target genes in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Values represent mean normalized mRNA expression (relative to housekeeping genes), shown for KO organoids relative to WT controls (dashed line). Data are presented as mean ± SD from three independent experiments. (F) Confocal imaging of Col ¾ and F-actin in ITGα2-WT and ITGα2-KO MMTV-PyMT organoids after one day in 3D Collagen I. (G, H) qPCR analysis of Vimentin and Slug mRNA expression in ITGα2-KO versus ITGα2-WT MMTV-PyMT organoids treated with Activin A (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graphs represent mean normalized expression values ± SD from four independent experiments. (I) Kaplan–Meier analysis correlating high vs. low mRNA expression of INHBA, ITGA2, ITGB1, and their combinations (ITGA2 + ITGB1, or INHBA + ITGA2 + ITGB1) with distant metastasis-free survival (DMFS) in patients with grade 3 breast cancer. Scale bars: 100 μm (A, C), 50 μm (A, zoom-in), 50 μm (F), 10 μm (F, zoom-in). P values: two-sided unpaired Mann–Whitney test (E), two-sided Kruskal-Wallis test with Dunn’s multiple comparisons (G, H), Log-rank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Imaging, Expressing, Cell Culture, Clone Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Integrin-TGFβ axis induces partial EMT in basal-like cells to lead collective invasion

doi: 10.1101/2025.04.04.647177

Figure Lengend Snippet: (A) DNA sequence of the Itgα2 gene to confirm gene knockout by Crispr-Cas9 gene editing. Red regions indicate the insertion of one base pair compared to the wildtype Itgα2 sequence. (B) Western blot analysis showing Itgα2 and GAPDH expression from whole cell lysates of MMTV-PyMT organoids WT or KO (gRNA1 or gRNA2). (C) Confocal imaging of Itgα2 and K8 in MMTV-PyMT organoids with Itgα2 WT or knockout (KO) grown in Collagen I for 3 days. White arrowheads: invading strands in Itgα2 WT organoids led by basal-like cells (low K8, high Itgα2, insets), White arrows: non-invading basal-like cells at the ECM interface in Itgα2 KO organoids (low K8, low Itgα2, insets). (D) Percentage of invasive organoids in MMTV-PyMT organoids Itgα2-WT versus Itgα2-KO (gRNA2). (E) Mean gray value of Col ¾ relative to Collagen I reflection in MMTV-PyMT organoids with Itgα2-WT and Itgα2-KO. Median: red lines, from n = 11 organoids per group from two independent experiments. (F) Single confocal slice showing INHBA and K8 expression in Itgα2-WT and Itgα2-KO MMTV-PyMT organoids cultured in 3D Collagen I for three days. Insets show basal-like cells (K8 low) guiding invasive strands (Itgα2-WT) or remaining at the organoid rim (Itgα2 KO, non-invasive). (G) Mean-gray value of Inhba in basal-like cells (K8-low) located at the rim of Itgα2 WT vs. KO MMTV-PyMT organoids (3D Collagen I, day 1). Median: red lines, n =11 cells from 6 Itgα2-WT organoids, and n = 12 cells from 7 Itgα2-KO organoids from one experiment. (H) qPCR analysis showing relative mRNA expression of CTGF in MMTV-PyMT Itgα2-KO organoids compared to Itgα2-WT organoids treated with Activin A ligand (20 ng/μl) or vehicle control (0.1% BSA) for three days. Bar graph represents mean normalized expression values (relative to housekeeping genes) ± SD from four independent experiments. (I) Kaplan-Meier plot correlating ITGB1 gene expression with DMFS in grade 3 breast cancer patients. Scale bars: 50 μm (C, F), 25 μm (C, F, Zoom in). P values, two-sided unpaired Mann–Whitney test (E, G), two-sided Kruskal-Wallis test (Dunn’s multiple comparison) (H), Logrank test (I).

Article Snippet: The following antibodies were used: rabbit anti-human Vimentin (Cat#ab92547, Abcam), chicken anti-human Vimentin (Cat#PA1-16759, Invitrogen) rabbit anti-human Keratin 14 (Cat# 905301, Biolegend), rat anti-mouse Keratin 8 (Cat# 531826, DSHB), rabbit anti-rat Collagen I cleavage site (Col ¾, Cat#0217-025, immunoGlobe), rabbit anti-human integrin α2 (Cat#ab181548, Abcam), rabbit anti-human Inhba (Cat#10651-1-AP, Proteintech), Mouse IgG1 isotype control (MAB002, R&D Systems), anti-Activin A antibody (Cat#AF338, R&D Systems).

Techniques: Sequencing, Gene Knockout, CRISPR, Western Blot, Expressing, Imaging, Knock-Out, Cell Culture, Control, Gene Expression, MANN-WHITNEY, Comparison