Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: FAPs accounted for the disparity in NMJ regeneration between MAS and TA muscles. (a) AChR and MyHC immunolabelling in myotubes differentiated from MuSCs isolated from MAS or TA muscles. Mature myotubes were treated with or without agrin (100 ng/mL) for 16 h. Scale bar = 50 μm. Graphs in the right panel show the quantifications of differentiation index (upper left), myotube width (upper right), AChR cluster counts normalized to myotube area (lower left), ratio of AChR/myotube area (lower middle) and Chrna1 expression quantified by real‐time quantitative PCR (lower right). n = 3. (b) AChR and MyHC staining of MuSCs single culture and cocultures with different FAPs. Scale bar = 50 μm. Graphs in the lower panel show (from left to right) the quantification of differentiation index, myotube area, AChR cluster density and ratio of AChR/myotube area. n = 3. (c) Schematic of the direct coculture of different FAPs with MAS‐derived MuSCs. (d) Representative images of tdTomato + FAPs and α‐BTX labelled AChRs (green) in recipient MAS and TA muscle. Scale bar = 20 μm. (e) Schematic of the FAPs transplantation assay. (f) Quantification of BTX signals per field. n = 6 mice/group. The data are shown as mean ± SD. Unpaired Student's t test was used in (a, upper right panel). Two‐way ANOVA followed by Tukey's post hoc test was used in (a, lower right panel and f). One‐way ANOVA followed by Tukey's post hoc test was used in (b). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Muscles, Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining, Derivative Assay, Transplantation Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: MSTN was the paracrine factor that distinguishes MAS‐derived FAPs from TA‐derived FAPs. (a) AChR and MyHC staining of MuSC single culture or cocultures with conditioned medium (CM) of MAS‐ or TA‐isolated FAPs. Scale bar = 50 μm. (b) Graphs show (from left to right) the quantification of myotube area, average AChR cluster size, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 6. (c) Venn diagram depicting the overlap among DEGs between 7 dpi MAS‐ and TA‐derived FAPs (orange), NMJ‐regulating genes (green) and genes encoding secreted proteins (blue). (d) Mstn expression levels were quantified by qPCR. n = 3. (e) Myostatin levels were quantified by ELISA in CM from 7 dpi MAS‐ or TA‐derived FAPs. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used in (b). Unpaired Student's t test was used in (d,e). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Derivative Assay, Staining, Isolation, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: Recombinant MSTN suppressed AChR clustering and miR‐206 expression. (a) AChR and MyHC staining of MuSCs cultures treated with or without recombinant MSTN (100 ng/mL) and follistatin (500 ng/mL). Scale bar = 50μm. (b) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. (c) The levels of miR‐206 expression in MuSCs cultures. Mature myotubes differentiated from MAS‐derived MuSCs were treated with control, agrin alone, agrin plus MSTN or agrin plus MSTN and follistatin. (d) AChR and MyHC staining of myotubes transfected with miR‐206 mimics or negative control (NC) prior to treatment with agrin and MSTN. Scale bar = 50 μm. (e) Graphs show (from left to right) the quantification of differentiation index, myotube area, AChR cluster counts normalized to myotube area and ratio of AChR/myotube area. n = 3. The data are shown as mean ± SD. One‐way ANOVA followed by Tukey's post hoc test was used. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Recombinant, Expressing, Staining, Derivative Assay, Control, Transfection, Negative Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Fibro‐Adipogenic Progenitors Regulate Orofacial Neuromuscular Junction Regeneration via Myostatin

doi: 10.1002/jcsm.70264

Figure Lengend Snippet: MSTN knockdown reversed the negative effect of MAS FAPs on in vitro AChR clustering. (a) MSTN protein and mRNA level in 7 dpi MAS FAPs transfected with negative control (pGPU6/GFP/Neo‐NC) or knockdown vectors (pGPU6/GFP/Neo‐shMSTN). (b) MSTN protein and mRNA level in 7 dpi TA FAPs transfected with negative control (pcDNA3.1(+)) or overexpression vectors (pcDNA3.1(+)‐MSTN). (c) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster counts normalized to myotube area (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN KD FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (d) Representative images of AChR and MyHC staining (left panel). Graphs in the right panel show the quantification of differentiation index (upper left), myotube area (upper right), AChR cluster density (lower left) and ratio of AChR/myotube area (lower right). CM was collected from NC and MSTN OE FAPs and added to myotubes differentiated from MAS‐derived MuSCs. Scale bar = 50 μm. n = 3. (e,f) Transcript levels of Chrna1 , Chrne , Chrng , Musk and Rapsn in myotubes treated with CM from NC or KD FAPs, and NC or OE FAPs. KD, knock down; OE, overexpression. The data are shown as mean ± SD. Unpaired Student's t test was used in (a–d). Two‐way ANOVA followed by Sidak post hoc test was used in (e,f). ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mature myotubes were treated with 100 ng/mL agrin (HY‐ P79236 , MedChemExpress, China) in DM or conditioned medium (CM) of 7 dpi FAPs supplemented with 2% HS for 16 h. For recombinant MSTN treatment, 100 ng/mL MSTN (HY‐ P72632 , MedChemExpress, China) with or without 500 ng/mL follistatin (HY‐ P70315 , MedChemExpress, China) was administered together with agrin treatment.

Techniques: Knockdown, In Vitro, Transfection, Negative Control, Over Expression, Staining, Derivative Assay