Figure 5 | Cell Death & Disease

Figure 5

From: Identification and characterization of PDGFRα+ mesenchymal progenitors in human skeletal muscle

Figure 5

Fibrogenic potential of PDGFRα+ cells and effects of TGF-β and PDGF signaling on PDGFRα+ cells. (a) Single PDGFRα+ cell-derived colonies were stained with antibodies against collagen I (Col1) and FABP4 or FABP4 and α-smooth muscle actin (α-SMA). FABP4 antibody labeled adipocytes with lipid droplet, whereas Col1 and α-SMA were detected only in fibrogenic cells. Scale bar: 50 μm. (b) CD56+ cells or PDGFRα+ cells were cultured with or without TGF-β1 (5 ng/ml) for 3 days, and the expressions of fibrosis-related genes were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Values are represented as the ratio to unstimulated CD56+ cells and shown as means±s.d. of three independent preparations. *P<0.01, **P<0.05. (c) Western blot analysis of collagen I, collagen III, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Results from two independent preparations were shown. (d) Matrix metalloproteinase (MMP) activity in the cell culture supernatants. Values are represented as the substrate amounts that were cleaved by MMPs contained in the supernatants and shown as means±s.d. of three independent preparations. (e) Tissue inhibitors of metalloproteinase-1 (TIMP-1) concentration in the cell culture supernatants. Values are shown as means±s.d. of three independent preparations. (f) PDGFRα+ cells were cultured in SFM with or without PDGF-AA (10 ng/ml) for 2 days. Inhibitors were added 1 h before PDGF-AA stimulation. The extent of PDGFRα phosphorylation is represented as the ratio to unstimulated control cells (cont) and shown as means±s.d. of three independent preparations. *P<0.01, **P<0.05. (g) Proliferation of PDGFRα+ cells was quantified by measuring BrdU incorporation. Values are represented as the ratio to unstimulated control cells (cont) and shown as means±s.d. of three independent preparations. *P<0.01; versus control culture. (h and i) PDGFRα+ cells cultured in SFM were stimulated with PDGF-AA (10 ng/ml) for 15 min. Inhibitors were added 1 h before PDGF-AA stimulation. Phosphorylation of Akt and p44/42 MAPK was assessed by immunoblotting (h) and immunofluorescent staining (i). Scale bar: 50 μm in i

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