Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 1. Cardiac fibrosis and innervation. (A) Masson’s trichrome staining of young (3-month-old) and old (10-month-old) WT and mdx mouse hearts. Scale bars, 100 μm. The graph shows the area as a percentage of the fibrosis index (fibrotic area/whole area). N = 3 biological replicates. (B) qPCR for fibrosis-related transcripts in 3- and 10-month-old mdx and WT hearts (Col1a1, Col3a1, Fn1, Tgfb1, Twist 1, Twist 2). N = 3 biological replicates. (C) Confocal images for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), MPs (F4/80), proteoglycan CSPG4, and fibronectin 1 (FN1) on myocardium sections of 3- and 10-month-old mice. The markers TH, SYN1, and CSPG4 are labeled in yellow, cardiac troponin (cTNNT) and F4/80 are in magenta, while FN1 is in white. Scale bars, 10 μm. Nuclei are counterstained with DAPI in blue. (D) Charts indicate positive area expressed as percentage of TH, SYN1, and F4/80 on whole area. N = 3 sections per N = 3 biological replicates. (E and F) Western blot analysis of TH, SYN1, and CSPG4 in mdx and WT hearts of young and old mice. Graphs show optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using Student’s t-test.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 2. MPs as main producers of CSPG4. (A) Graphic illustration of CSPG4-CAR-Ts cells against MPs expressing CSPG4. Created with Biorender.com. Diagrams quantifying the percentage of live cells of bone marrow-derived MPs of both WT and mdx M0 (nonactivated), M1 (pro-inflammatory) and M2 (anti-inflammatory), or CFs after exposure to CSPG4.CARTs compared to cells co-cultured with control CART (cCART). N = 3 biological replicates. (B) ELISA assay for CSPG4 in supernatants (SUP) isolated from WT and mdx MPs. The chart illustrates the optical density (OD) of CSPG4 in the two groups. Each dot represents the eluted fractions, N = 16. (C) FACSymphony of cardiac tissues. The first chart represents a scatter plot of t-SNE relative to concatenated total cells from 3- or 10-month-old WT and mdx hearts. The second chart illustrates the distribution of CD45neg cells (light pink cluster) and myeloid cells (dark pink cluster) in all groups. (D) Graph of FACS analysis relative to number of total MPs (F4/80) positive for CSPG4 in 3- or 10-month-old WT and mdx hearts. (E) Expression of CSPG4 in subpopulations of MPs: CD80pos (M1), CD206 pos (M2), Ly6Clow (resident), and Ly6Chigh (monocytes) in 3- or 10-month-old WT and mdx hearts. Error bars show SEM. *p < 0.05, **p < 0.01 calculated using Student’s t-test and one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Expressing, Derivative Assay, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 3. MPs release CSPG4 in media and inhibit axon growth of dorsal root ganglia. (A) Rendering of 3D NCJ generation. The picture was created with Biorender.com and illustrates the composition of the 3D NCJ model: WT dorsal root ganglia (DRG), WT cardiomyocytes (CMs), and WT CFs were encapsulated in PEG-FB hydrogel conditioned with the supernatants derived from MP cultures of WT, WT + CSPG4 blocking antibody (BAb), mdx, and mdx + CSPG4 (BAb). (B) Brightfield images of CFs, CMs, and DRG after 1 day of culture (upper panel) and 3D constructs of NCJ models after 7 days (lower panel). Scale bars, 100 μm. (C) Confocal images of 3D NCJ models: WT, WT + CSPG4 BAb, mdx, mdx + CSPG4 BAb. DRG are labeled with tubulin beta 3 (TUBB3, in yellow), CMs with cardiac troponin (cTNNT, in magenta), and CFs with fibroblast marker (ER- TR7, in white). (D) The graph indicates the area (%) occupied by neuron endings labeled by TUBB3 inside the 3D NCJ models. N = 3 biological replicates, N = 2 sections/constructs. Scale bars, 10 μm. **p < 0.01, ***p < 0.001 were determined using one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Derivative Assay, Blocking Assay, Construct, Labeling, Marker

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 4. Cardiac innervation in old mdx and WT mice. (A) Immunofluorescence staining for tyrosine hydroxylase (TH), Synapsin 1 (SYN1), total MPs (F4/80) and proteoglycan (CSPG4), fibronectin 1 (FN1) on cardiac sections from 10-month-old mice. The markers TH and SYN1 are labeled in yellow, while cardiac troponin (cTNNT) and F4/80 are in magenta, FN1 in white, and CSPG4 in yellow or white. DAPI nuclear counterstaining is in blue. Scale bars, 10 μm. (B) Charts indicate area expressed as percentage of TH, SYN1, and F4/80 (positive area/whole area). N = 3 sections per N = 3 biological replicates. (C and D) Western blot analysis for TH, SYN1, and CSPG4 quantified as optical density (OD) of protein bands normalized to vinculin (VCL). N = 3 biological replicates. (E) qPCR relative to markers for neurotransmitter production (Th), survival (Ngf, Ntrk1, and Ngfr) and axon guidance (Tubb3) in DRG. N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using one-way ANOVA.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Staining, Labeling, Western Blot

Journal: The Journal of pathology

Article Title: Macrophages producing chondroitin sulfate proteoglycan-4 induce neuro-cardiac junction impairment in Duchenne muscular dystrophy.

doi: 10.1002/path.6362

Figure Lengend Snippet: Figure 5. Histological analysis and gene profile of cardiac tissue after treatment with givinostat in old mdx and WT mice. (A) Confocal images of immunostaining for gap junctions (CX43) and cardiomyocytes positive for cardiac troponin (cTNNT), N = 3 sections per N = 3 biological replicates. Scale bars, 50 μm. (B) Charts indicate area as percentage of CX43 (positive area/whole area). (C and D) Representative Masson’s trichrome images of WT, mdx saline, and mdx Giv hearts and fibrosis index quantification (fibrotic area/whole area) after 60 days of treatment. Scale bars, 100 μm. N = 3 biological replicates. (E) qPCR of fibrosis-related genes (Tgfb1, Col1a1, Col3a1, Twist1, Twist2, Fn1), cell surface proteoglycan (Cspg4); the enzyme catalyzes the transfer of sulfate groups on CSPG4 chains (Chst11) and inflammatory markers (Adgre1 and Mmp9). N = 3 biological replicates. Error bars show SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were calculated using one-way ANOVA and Student’s t-test.

Article Snippet: Western blot Proteins were extracted from tissues using lysis buffer (50 mM TRIS HCl pH 7.5), 0.6 M sucrose (Catalogue No. S0389, Sigma-Aldrich), 50% glycerol (Catalogue No. G5516, Sigma-Aldrich), 1% TRITON, 50 mM NaCl, 10 mM NaF, 2 mM sodium orthovanadate (Na3VO4, Catalogue No. 450243 Sigma-Aldrich), 1 mM PMSF (Catalogue No. 36978, Thermo Scientific,Waltham,MA, USA), 5 mM β-glycerophosphate (Catalogue No. G9422, Sigma-Aldrich), 1000X protease inhibitors (Catalogue No. 04693116001, Roche) for 30 min on ice, sonicated, and centrifuged at 12,000 g for 15 min at 4 C. Proteins were resolved using SDS-PAGE, transferred to nitrocellulose membranes (Catalogue No. GE10600079, Amersham Protran, Johns Creek, GA, USA), blocked with 5% nonfat dry milk powder (Catalogue No. 1.15363, Merck Millipore, Billerica, MA, USA) in PBS 1 for 45 min, and incubated at 4 C overnight with primary antibodies: tyrosine hydroxylase (1:1000, ab152), Synapsin 1 (1:1000, Cell Signaling Technology, #5297), CSPG4 (1:1000, Cell Signaling Technology, #52635), and Vinculin (1:500, V9131, Sigma-Aldrich), followed by incubation with secondary antibodies (antiRabbit NA934AV, 1:5000; anti-Mouse LNXa931/AE, 1:10000, GE Healthcare Life Sciences, Havelock, NE, USA) for 1 h at RT.

Techniques: Immunostaining, Saline