Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, qRT-PCR analysis of Tgfb1, 2 and 3 transcripts expression during in vitro differentiation of primary muscle cells shows different profiles. b, qRT-PCR analysis of Alk5 and Tgfbr2 transcripts expression describes a constant expression of the receptors during primary muscle cells differentiation. c, qRT-PCR analysis of the TGFβ target gene Smad7 transcript expression reveals a decreased activity of the pathway alongside in vitro primary muscle cell differentiation. d, Phospho-SMAD2/3 immunofluorescent staining of proliferating, differentiating and differentiated primary myoblasts reveals a constant and basal activation of the pathway. e, Phospho-SMAD2/3 and SMAD2/3 western-blot analysis of proliferating, differentiating and differentiated primary myoblasts confirms a decrease in SMAD2/3 phosphorylation during differentiation. f, qRT-PCR analysis of Tgfb1, 2 and 3 transcripts expression during muscle tissue regeneration induced by CTX injection shows specific expression profiles. g, Immunofluorescent staining for phospho-SMAD3 on 4-days regenerating TA muscles cryosections confirms the presence of active TGFβ signaling in the tissue. Arrowheads indicate phospho-SMAD3 + nuclei within myofibres. Scale bars: e, 200μm. g, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments.

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Quantitative RT-PCR, Expressing, In Vitro, Activity Assay, Cell Differentiation, Staining, Activation Assay, Western Blot, Injection, Muscles

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Experimental scheme. Primary myoblasts were induced to differentiate in medium containing TGFβ recombinant proteins. b, Immunofluorescent staining for Pan-MyHC of 3-days differentiated myotubes. c, qRT-PCR analysis for Myh3 (Embryonic Myosin Heavy Chain) transcript expression by of 3-days differentiated primary myoblasts indicates that stimulation of the pathway downregulates its expression compared to the control. d, Percentage of Pan-MyHC-expressing cells of 3-days differentiated primary myoblasts. e, Fusion index of 3-days differentiated primary myoblasts shows that TGFβ stimulation inhibits fusion. Scale bars: c, 1000μm. Data are presented as mean ± s.e.m. from at least three independent experiments. N.D.=Not significant, compared with Control (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Recombinant, Staining, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Experimental scheme. Primary myoblasts seeded at low density (5,000 cells/cm2) were differentiated for two days, split and re-plated at high density (75,000 cells/cm2) and cultured for two more days. b, Immunofluorescent staining for MYOGENIN of primary muscle cells pre-differentiated for 48h and re-plated at high density, confirms that more than 90% of cells express Myogenin. c, Immunofluorescent staining for the Myosin Heavy Chain isoforms (Pan-MyHC) of re-plated primary myotubes cultured for 48 hours. d, Percentage of Pan-MyHC-expressing cells of re-plated myotubes shows that cells were differentiated in all conditions. e, Fusion index of re-plated myotubes reveals that TGFβ stimulation inhibits fusion. f, Percentage of nuclei in the smallest and largest myotube classes. TGFβ-treated myotubes are characterized by less nuclei per myotube. Scale bars: b, 400μm c, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments. ** P <0.01, *** P <0.001, N.D.=Not significant, compared with Control (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Cell Culture, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Primary myoblasts were treated with TGFβ1, 2 or 3 for 24h and incubated with BrdU for the last 40 minutes before fixation. 0uantification of BrdU + cells shows no differences. b, Primary myoblasts were treated with TGFβ1, 2 or 3 for 24h in proliferating or differentiating conditions. TUNEL + cells were quantified, and no particular death rates were detected. c, Primary myoblasts were treated with TGFβ1, 2 or 3 for 24h in proliferating condition. When treated, cell layer was scratched and washed with PBS. Scratch-wound images were taken after 24 hours of treatment. Cells were stained with NucBlue. The quantification of nuclei within the scratch-wound reveals that motility is not affected. Scale bars: b, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments. *** P <0.001, compared with Control (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Incubation, TUNEL Assay, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, qRT-PCR analysis of TGFβ target genes transcript expression in primary myocites treated with TGFB1 protein or ITD-1 compound proves that Smad7 and Klfl0 are over-expressed when the signaling pathway is activated and inhibited when TGFβ cascade is blocked, b, Nuclear phospho-SMAD2/3 and SMAD2/3 western blot analysis of primary myoblast treated with TGFB1 protein, ITD-1 compound or both combined. The intracellular mediators SMAD2/3 are phosphorylated upon TGFβ stimulation, while ITD-1 is able to reduce their phosphorylation. c, Immunofluorescent staining for Pan-MyHC of re-plated primary myotubes cultured for 48 hours. d, Aggregation index of re-plated myotubes shows that ITD-1 treatment leads to the formation of myotubes with higher numbers of nuclei compared to the control. e, Fusion index of re-plated myotubes confirms the enhanced fusion when TGFβ cascade is inhibited. Quatntification of the diameter of re-plated myotubes ( f ) and of the distribution of branched-myotubes ( g ) of re-plated cells highlight aberrant morphology of syncitia treated with ITD-1. Scale bars: c, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments. * P <0.05, *** P <0.Q01, compared with Control (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Experimental scheme. H2B-GFP primary myoblasts were seeded at low density (5000 cells per cm2), treated with TGFβ1 protein or ITD-1 compound, stained with SiR-Actin and differentiated for 2 days. Membrane-TdTomato primary myoblasts were seeded at low density (5000 cells per cm 2 ) and differentiated for two days. Both populations were split and co-cultured (50/50) at high density (50,000 cells/cm 2 ) and cultured for two more days. In the last 40 hours, cells were recorded live by confocal microscopy. b, Live-imaging frames of co-cultured pre-differentiated myoblasts confirm the phenotype previously observed. TGFβ activation inhibits fusion, ITD-1 enhance fusion. c, Quantification of H2B-GFP nuclei within TdTomato myotubes. d, Quantification of heterologous myotubes (double positive for SiR-Actin and TdTomato). e, Quantification of Myotube-to-Myotube events. ITD-1 treatment allows more myotube-to-myotube events compared to the control. Scale bars: b, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, compared with Control (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Staining, Membrane, Cell Culture, Confocal Microscopy, Imaging, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Experimental scheme. Adult mice TA muscles were subjected to CTX injury and regenerating tissues were injected with either TGFβl proteins or ITD-1 compound 3 days after damage. b, Immunofluorescent staining for LAMININ of 7-days regenerating TA muscles. c, Quantification of myofiber size (cross sectional area, CSA). While the injection of TGFβ strongly reduces the fibers size, ITD-1 administration increases the CSA. d, Distribution of myofiber CSA. e, Distribution of myonuclei per fiber shows that the inhibition of TGFβ cascade leads to the formation of multi nucleated myofibers, while TGFβ activation reduces the number of myonuclei per fibers. f, Experimental scheme. Adult mice TA muscles were subjected to CTX injury and regenerating tissues were injected with either TGFβ proteins or ITD-1 compound 3, 6 and 9 days after damage. 14 days after injury, force measurements were performed and TA muscles collected. g, Immunofluorescent staining for LAMININ of 14-days regenerating TA muscles. h, Quantification of myofiber size confirms the phenotypes observed at 7d.p.i.. i, Distribution of myofiber CSA. j, Specific force measurement of regenerating muscles. While TGFβ1-treated muscles are weaker compare to the control, ITD-1 injected muscles are funstional. Scale bars: b, g , 100μm. Data are presented as mean ± s.e.m. from at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, N.D.=Not significant, compared with Control (Unpaired two-tailed Student’s t-test). Control represents mock-treated contralateral tibialis anterior muscle.

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Muscles, Injection, Staining, Inhibition, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: Transcriptomic analysis was performed on differentiated myocytes treated with either TGFBI or ITD1. a, Venn Diagram showing differentially expressed gene overlap across the three conditions. b, Heatmaps of TGFβ Target genes, myogenic genes and fusion genes. c, Volcano Plot showing the Ingenuity Pathway Analysis (IPA). Among the top modulated pathways, Actin Signaling Pathway is highlighted. d, Phalloidin staining of 1-day differentiated myocytes. These pictures were analyzed with OrientationJ (ImageJ Plug-in) to obtain a color-coded orientation mask. e, Average cell spread quantification. TGFpi treatment reduces cell size; ITD-1 promotes cell spreading. f, Quantification of orientation coherency of the Actin fibers. Both treatments reduce coherency compare to the control. g, Immunofluorescent staining for all the Mysosin Heavy Chain isoforms (Pan-MyHC) of re-plated primary myotubes cultured for 48 hours with ITD-1, Latrunculin, or both. h, Fusion index of re-plated myotubes shows that Latrunculin significantly reduces the parameter when administrated. i, Percentage of nuclei in the smallest myotube classes. ITD-1-treated myotubes are characterized by less nuclei per myotube, while Latrunculin increases the percentage of nuclei in small myotubes when administrated alone or together with ITD-1. j, Percentage of nuclei in the biggest myotube classes. ITD-1 strongly increases the number of nuclei in big myotubes, but Latrunculin blunts this effect, reducing the percentage. Scale bars: d, 40μm. g, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments. Coherency was calculated from at least 150 cells per condition. * P <0.05, ** P <0.01, *** P <0.001, compared with Control. # P <0.05, ## P <0.01, ### P <0.001, compared with ITD-1 (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Staining, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Representative scheme of the Actin signaling pathway genes modulated by TGFβ cascade. b, Live-imaging frames of pre-differentiated myocytes expressing H2B-GFP and stained with SiR-Actin. Arrowheads indicate fusion events, arrow depicts cell-cell interaction. In control condition, fusion occurs linearly, while ITD-1 treatment allows perpendicular fusion. On the other hand, TGFβ1 allows cell-cell interactions, but blocks fusion. Scale bars: b, 200μm. Data are presented as mean ± s.e.m. from at least three independent experiments.

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Imaging, Expressing, Staining

Journal: bioRxiv

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1101/557009

Figure Lengend Snippet: a, Experimental scheme. A dox-Inducible Myomaker- and Myomerger-expressing fibroblasts were used to test TGFβ1 effect on cell-cell fusion. Dox was administrated at day 0 and refreshed every day, while TGFβ1 either at day 0, 1, 2 or 3. b, Aggregation index of 4-days Myomaker and Myomerger-expressing fibroblasts showing no significant reduction of the fusion process when TGFβ1 is administrated compared to the control. c, GFP-myomaker-infected fibroblasts, transduced with dox-inducible myomerger, were visualized with fluorescent microscopy. TGFβ stimulation does not reduce the fusion process. Scale bars: c, 400μm. Data are presented as mean ± s.e.m. from at least three independent experiments. * P <0.05 compared with Control (Unpaired two-tailed Student’s t-test).

Article Snippet: Recombinant mouse TGFβ1 (R&D Systems) was diluted in saline and 250 ng (25 ul) was injected into the TA every injection.

Techniques: Expressing, Infection, Transduction, Microscopy, Two Tailed Test