Fig 2: Pdgfrb-Cre effectively targets recombination in quiescent PDGFRß+ cells and activated myofibroblasts. a Immunofluorescence micrographs of skeletal muscle from mTmG;Pdgfrb-Cre mice co-stained with anti-PDGFRß antibody. Scale bar 10 µm. b, c Quantification of GFP reporting and PDGFRß antibody colocalisation in skeletal muscle (n = 7). d Flow cytometric analysis of PDGFRß expression in GFP+ cells sorted from mTmG:Pdgfrb-Cre mouse skeletal muscle (n = 3). e Immunofluorescence micrographs of skeletal muscle sections harvested from mTmG;PDGFR ß-Cre reporter mice 4, 8 and 21 days after control (PBS) or CTX intramuscular injection. Scale bars 30 µm. f Percentage field coverage of GFP+ cells at 4, 8, 21 and 60 days after control (PBS) or CTX intramuscular injection (n = 4). g Quantitation of GFP+ nuclei at 4, 8, 21 and 60 days after control (PBS) or CTX intramuscular injection (n = 4). h Gene expression profile of freshly sorted GFP+ cells from skeletal muscle at day 10 following control (PBS) or CTX intramuscular injection (n = 4). i Immunofluorescence staining of a typical GFP+ cell sorted from uninjured skeletal muscle of mTmG;Pdgfrb-Cre reporter mice and plated on tissue culture plastic for 5 days. Scale bar 50 µm. j qPCR analysis of the genes encoding PDGFRß, aSMA, Col1A1 and TIMP1 in freshly sorted GFP+ cells from mTmG;Pdgfrb-Cre reporter mice and from GFP+ cells cultured for 7 and 14 days (n = 4). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t-test)

Fig 3: av integrins on PDGFRß+ cells regulate skeletal muscle fibrosis via TGFß activation. a av expression on PDGFRß+ cells from control and Itgav flox/flox;Pdgfrb-Cre (av Cre) mice using flow cytometry (n = 3). b av Cre and control mice received intramuscular CTX with muscles harvested at day 21. c Representative images of picrosirius red stained sections 21 days after CTX. Upper panels: scale bar 25 µm, Lower panels: 400 µm. d Digital image analysis quantification of picrosirius red staining in control and av Cre mice day 21 post CTX or control injection (n = 7). e qPCR of Col1a1 in control and av Cre PDGFRß+ cells culture-activated for 5 days (n = 7). f qPCR of TGFß1 in control and av Cre PDGFRß+ cells culture-activated for 5 days (n = 7). g qPCR of Col1a1 in GFP+ cells from mTmG;Pdgfrb-Cre mice cultured with CWHM 12 and control for 5 days (n = 3). h TGFß activation by control- or CWHM 12-treated GFP+ cells from mTmG;Pdgfrb-Cre mice (n = 6). TGFß activation was assessed alone, with TGFß–blocking antibody (Anti–TGFß), and with recombinant human TGFß1 (rhTGFß). i Representative images of picrosirius red stained sections from mice treated with CWHM12 or control from the time of CTX. Mice received rhTGFß1 (lower panels) to the region of injured muscle every 72 h up to 21 days. j Digital image analysis quantitation of picrosirius red stained sections from CWHM12 or control treated mice that received rhTGFß1 or PBS to the region of injured muscle (n = 5). k Representative images of phospho-SMAD 3 stained sections from control or CWHM12-treated mice. Scale bar 30 µm. l Quantitation of phospho-SMAD 3 stained sections from mice treated with control or CWHM12 from the time of CTX (n = 10). m Force required for passive elongation of muscles 21 days following intramuscular CTX in mice treated with CWHM 12 or control (n = 9). n Laceration-induced skeletal muscle fibrosis model dosing regime. o Representative images of picrosirius red stained sections from control and CWHM 12 treated mice. Scale bar 25 µm. p Digital image analysis quantitation of picrosirius red staining in CWHM12 and control treated mice (n = 10). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 (Student’s t-test)