Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

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. N = 3 biologically independent experiments for each time point. b qRT-PCR analysis of Alk5 and Tgfbr2 transcript expressions describes a constant expression of the receptors during primary muscle cell differentiation. N = 6 biologically independent experiments for each time point. 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. N = 3 biologically independent experiments for each time point. d p-SMAD2/3 immunofluorescent staining of proliferating, differentiating, and differentiated primary myoblasts reveals a constant and basal activation of the pathway. N = 3 primary cell cultures. e p-SMAD2/3 and SMAD2/3 western-blot analysis of proliferating, differentiating, and differentiated primary myoblasts confirms a decrease in SMAD2/3 phosphorylation during differentiation. N = 3 biologically independent experiments. Scale bars: d 200 μm. Data are presented as mean ± SEM. Source data are provided as a Source Data file.

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

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

Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

Figure Lengend Snippet: a Experimental scheme. Primary myoblasts seeded at low density (5000 cells/cm 2 ) were differentiated for 2 days, split, and re-plated at high density (75,000 cells/cm 2 ) and cultured for 2 more days. b Immunofluorescent staining for MYOGENIN of primary myocytes pre-differentiated for 48 h and re-plated at high density confirms that >90% of cells express Myogenin. N = 7 biologically independent experiments. c qRT-PCR analysis for Myogenin and Smad7 transcript expression of re-plated primary myocytes cultured for 24 h with or without TGFβ1 recombinant protein. Although TGFβ1 stimulation activates Smad7 expression, it does not affect Myogenin transcript levels. N = 6 primary cell cultures. d Immunofluorescent staining for the MYOSIN HEAVY-CHAIN isoforms (Pan-MyHC) of re-plated primary myocytes cultured for 48 h. e Percentage of Pan-MyHC-expressing cells of re-plated myotubes shows that cells were differentiated in all conditions. N = 5 biologically independent experiments. f Fusion index of re-plated myotubes reveals that TGFβ stimulation inhibits fusion. N = 5 biologically independent experiments. g Percentage of nuclei in the smallest and largest myotube classes. TGFβ-treated myotubes are characterized by less nuclei per myotube. N = 11 (control) and 15 (TGFβ1) biologically independent experiments. Scale bars: b 400 μm; d 200 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. ** and *** denote a significant difference with the Control group of P < 0.01 and P < 0.001, respectively. NS not significant. Source data are provided as a Source Data file.

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

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

Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

Figure Lengend Snippet: a Experimental scheme. Primary myoblasts seeded at 10,000 cells/cm 2 were induced to differentiate with or without TGFβ1 recombinant protein. After 12 h, cells were recorded live for 12 h during early differentiation and for 24 h during late differentiation. b Quantification of the percentage of MYOD1+ nuclei of primary myocytes cultured for 48 h with or without TGFβ1 recombinant protein. N = 3 biologically independent experiments. c qRT-PCR analysis for Myogenin transcript expression of primary myotubes cultured for 60 h with or without TGFβ1 recombinant protein. N = 5 biologically independent experiments. d Brightfield live-imaging frames of differentiating myoblasts confirm that TGFβ stimulation reduces fusion. N = 6 primary cell cultures. e Early differentiation movies were used to quantify cell speed, covered distance and cell–cell contact frequency. None of these parameters were significantly different between control and TGFβ1-treated myoblasts. N = 240 cells for each condition examined over six biologically independent experiments. f Late differentiation movies were used to quantify cell–cell contact frequency, the percentage of cell–cell contacts that result in fusion events and the percentage of cells that make contacts but do not fuse. Although TGFβ1 stimulation does not impair cell–cell contact frequency, many TGFβ1-treated contacting myoblasts make contacts but do not accomplish fusion. N = 240 cells for each condition examined over six biologically independent experiments. Scale bars: d 400 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. *** denote a significant difference with the Control group of P < 0.0001. NS not significant. Source data are provided as a Source Data file.

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

Techniques: Recombinant, Cell Culture, Quantitative RT-PCR, Expressing, Imaging, Control, Two Tailed Test

Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

Figure Lengend Snippet: a qRT-PCR analysis of TGFβ target genes transcript expression in primary myocytes treated with TGFβ1 protein or ITD-1 compound proves that Smad7 and Klf10 are over-expressed when the signaling pathway is activated and inhibited when TGFβ cascade is blocked. N = 10 (Control), 8 (TGFβ1), and 8 (ITD-1) biologically independent experiments. b Nuclear p-SMAD2/3 and SMAD2/3 western blot analysis of primary myoblast treated with TGFβ1 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. N = 3 biologically independent experiments. c Immunofluorescent staining for Pan-MyHC of re-plated myocytes cultured for 48 h. N = 8 primary cell cultures. d Aggregation index of re-plated myocytes shows that ITD-1 treatment leads to the formation of myotubes with higher numbers of nuclei compared to the control. N = 8 biologically independent experiments. e Fusion index of re-plated myocytes confirms the enhanced fusion when TGFβ cascade is inhibited. N = 8 biologically independent experiments. Diameter of re-plated myotubes ( f ) and of the distribution of branched-myotubes ( g ) of re-plated cells highlight aberrant morphology of syncytia treated with ITD-1 N = 4 ( f ) and 6 ( g ) biologically independent experiments. Scale bars: c 200 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. * and *** denote a significant difference with Control group of P < 0.05 and P < 0.001, respectively. Source data are provided as a Source Data file.

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

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

Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

Figure Lengend Snippet: a Experimental scheme. H2B-GFP primary myoblasts were seeded at low density (5000 cells/cm 2 ), treated with TGFβ1 protein or ITD-1 compound, stained with SiR-Actin, and differentiated for 2 days. Membrane-tdTOMATO primary myoblasts seeded at low density (5000 cells/cm 2 ) and were differentiated for 2 days. Both populations were split and co-cultured (50/50) at high density (75,000 cells/cm 2 ) for 2 more days. In the last 40 h, cells were recorded live by confocal microscopy. b Live-imaging frames of co-cultured pre-differentiated myocytes confirm the phenotype previously observed. TGFβ activation inhibits fusion, while ITD-1 enhance fusion. N = 8 biologically independent primary co-cultures. c Quantification of H2B-GFP nuclei within tdTOMATO myotubes. N = 8 biologically independent experiments. d Quantification of heterologous myotubes (double positive for SiR-Actin and tdTOMATO). N = 6 biologically independent experiments. e Quantification of Myotube-to-Myotube events. ITD-1 treatment allows more myotube-to-myotube events compared to the control. N = 6 biologically independent experiments. Scale bars: b 200 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. *, **, and *** denote a significant difference with the Control group of P < 0.05, P < 0.01, and P < 0.001 respectively. Source data are provided as a Source Data file.

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

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

Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

Figure Lengend Snippet: a Experimental scheme. Adult murine tibialis anterior (TA) muscles were subjected to CTX injury and regenerating tissues were injected intramuscularly with either TGFβ1 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 fibers size, ITD-1 administration increases fibers size. d Distribution of myofiber CSA. N = 10 (Control), 5 (TGFβ1), and 5 (ITD-1) biologically independent TA muscles. e Distribution of myonuclei per fiber shows that the inhibition of TGFβ cascade leads to the formation of multinucleated myofibers, while TGFβ activation reduces the number of myonuclei per fibers. N = 6 (Control), 3 (TGFβ1), and 3 (ITD-1) biologically independent TA muscles. f Experimental scheme. Adult murine 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. Fourteen days after injury, force measurements were performed, and TA muscles were collected. g Immunofluorescent staining for LAMININ of 14-day regenerating TA muscles. h Quantification of myofiber size confirms the phenotypes observed at 7 d.p.i. N = 8 (Control), 4 (TGFβ1), and 4 (ITD-1) biologically independent TA muscles. i Distribution of myofiber CSA. N = 8 (Control), 4 (TGFβ1), and 4 (ITD-1) biologically independent TA muscles. j Specific force measurement of regenerating muscles. While TGFβ1-treated muscles are weaker compared to the control, ITD-1-injected muscles show no differences. N = 18 (Control), 9 (TGFβ1), and 9 (ITD-1) biologically independent TA muscles. Scale bars: b , g , 100 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. *, **, and *** denote a significant difference with control group of P < 0.05, P < 0.01, and P < 0.001, respectively. Control represents mock-treated contralateral TA muscle. Source data are provided as a Source Data file.

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

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

Journal: Nature Communications

Article Title: TGFβ signaling curbs cell fusion and muscle regeneration

doi: 10.1038/s41467-020-20289-8

Figure Lengend Snippet: Transcriptomic analysis was performed on differentiated myocytes treated with either TGFβ1 or ITD1. N = 3 biologically independent experiments. a Heatmaps of TGFβ target genes, myogenic genes, and fusion genes. b Volcano plot showing the Ingenuity Pathway Analysis (IPA). Among the top modulated pathways, Actin Signaling Pathway is highlighted. c Phalloidin staining of 1-day differentiated myocytes. These pictures were analyzed with OrientationJ (ImageJ Plug-in) to obtain a color-coded orientation mask. N = 8 primary cell cultures. d Average cell spread quantification. TGFβ1 treatment reduces cell size; ITD-1 promotes cell spreading. N = 8 primary cell cultures. e Quantification of orientation coherency of the actin fibers. Both treatments reduce coherency compared to the control. N = 150 cells for each condition examined over six biologically independent experiments. f Quantification of cell–cell contact frequency. N = 240 cells for each condition examined over six biologically independent experiments. g Immunofluorescent staining for Pan-MyHC of re-plated primary myotubes cultured for 48 h with ITD-1, Latrunculin, or both. N = 5 primary cell cultures. h Fusion index of re-plated myotubes shows that Latrunculin significantly reduces the parameter when administrated. N = 5 biologically independent experiments. i Percentage of nuclei in the smallest myotube classes. ITD-1-treated myotubes are characterized by a lower number of nuclei in the smallest myotubes, while Latrunculin increases the percentage of nuclei in small myotubes when administrated alone or together with ITD-1. N = 5 biologically independent experiments. 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. N = 5 biologically independent experiments. Scale bars: c 40 μm; g 200 μm. Data are presented as mean ± SEM. Unpaired two-tailed Student’s t tests were used to compare between data. *, **, and *** denote a significant difference with the Control group of P < 0.05, P < 0.01, and P < 0.001 respectively. # , ## , and ### denote a significant difference with ITD-1 group of P < 0.05, P < 0.01, and P < 0.001, respectively. NS not significant. Source data are provided as a Source Data file.

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

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

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Recombinant sTLR proteins are monomeric, properly folded, and biologically functional. A, 100 μg of each purified sTLR protein as indicated was loaded for 7.5% SDS-PAGE and stained with Coomassie blue dye. B, each purified sTLR protein as indicated was analyzed by size exclusion chromatography using a Superdex 200 column. C, soluble TLRs were incubated in microtiter plate wells coated with either the anti-TLR1 mAb (clone GD2F4) or the anti-TLR2 mAb (T2.5) as indicated. Binding of soluble TLR1, TLR1P315L, or TLR2 was detected using HRP-conjugated anti-HA and anti-FLAG mAbs. D, SW620 cells were co-transfected with full-length TLR1, TLR2, an IL-8 promoter-driven luciferase reporter gene, and a Renilla luciferase transfection control. 48 h post-transfection, the cells were stimulated with 10 ng/ml Pam3CSK4 with or without 1 μg/ml soluble TLR1 or TLR2 as indicated. Firefly luciferase activities were normalized to that of the Renilla luciferase control. These values were normalized to that of empty CMV vector whose value was taken as 1. Error bars represent the S.D. of three independent events. mAu, milliabsorbance units.

Article Snippet: Cells were co-transfected with 50 ng each of TLR1 and TLR2 together with 75 ng of a firefly luciferase reporter gene driven by an NF-κB promoter and 25 ng of a Renilla luciferase (pRL-null) transfection control (Promega).

Techniques: Recombinant, Functional Assay, Purification, SDS Page, Staining, Size-exclusion Chromatography, Incubation, Binding Assay, Transfection, Luciferase, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex *

doi: 10.1074/jbc.M113.453266

Figure Lengend Snippet: Either LBP or soluble CD14 enhances cellular responses to Pam3CSK4 and OspA. HEK 293F cells were co-transfected with vectors expressing full-length TLR1 and TLR2 or empty CMV control vector as indicated together with an NF-κB-promoter driven luciferase reporter gene and a Renilla luciferase reporter gene. About 48 h post-transfection, cells were stimulated with 1 ng/ml Pam3CSK4, OspA, or the non-acylated Ac2CSK4 control in the presence of 0.1 μg/ml LBP, sCD14, or human serum albumin (HSA) as indicated (left side). In one set of experiments, agonists were preincubated with proteins for 1 h at 37 °C prior to addition to transfected cells (right side). Cell values on the y axis represent the level of constitutive reporter activation normalized to the empty CMV vector control (value of 1). Error bars represent the S.D. of three independent values.

Article Snippet: Cells were co-transfected with 50 ng each of TLR1 and TLR2 together with 75 ng of a firefly luciferase reporter gene driven by an NF-κB promoter and 25 ng of a Renilla luciferase (pRL-null) transfection control (Promega).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Activation Assay