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. 2015 Jun 29;212(7):1061–1080. doi: 10.1084/jem.20141601

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© 2015 Lee et al.

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Figure 7. — PTD-DBM induces collagen production in human dermal fibroblasts. (A) PTD-DBM is a synthetic peptide that includes a PTD for enhanced protein delivery, a linker for flexibility, DBM, and lysine conjugated with FITC for visualization. (B) Human dermal fibroblasts (n = 2–3 cells), which were treated with 2 or 10 µM PTD-DBM for 2 d, were analyzed using Western blotting to detect β-catenin, α-SMA, collagen I, endothelin-1, c-Myc, cyclin D1, and α-tubulin. Endothelin-1 and GAPDH expression were measured by RT-PCR analysis (n = 2 independent experiments). Relative densitometry values are shown below the blots as ratios relative to the levels of loading control (α-tubulin or GAPDH). (C) Human dermal fibroblasts, treated with 2 or 10 µM PTD-DBM for 2 d, were subjected to ICC analyses to detect β-catenin or collagen I. Representative ICC images are shown (top), and mean intensity values are presented (bottom; *, P < 0.05; **, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (D) Human dermal fibroblasts were treated with PTD-DBM for 2 d after treatment with β-catenin siRNA (β-catenin si). Western blot analyses of WCLs were performed with antibodies against β-catenin, α-SMA, collagen I, endothelin-1, or α-tubulin (n = 2 independent experiments). (E) Human dermal fibroblasts, treated with 2 µM PTD-DBM and/or 10 µM bosentan for 2 d, were analyzed by Western blotting to detect β-catenin, α-SMA, collagen I, and α-tubulin (n = 2 independent experiments). (D and E) Relative densitometry values are shown below the blots. (F) Wnt reporter activity (left), Col1a2 reporter activity (middle), and the concentration (right) of collagen in the supernatants of human dermal fibroblasts were measured after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). (G) A collagen gel contraction assay after treatment with 2 or 10 µM PTD-DBM, 50 ng/ml Wnt3a, or 10 ng/ml TGF-β was performed and representative images are shown (left). Gel weight quantitation is shown (right; ***, P < 0.0005; n = 3 independent experiments). (H) An in vitro wound healing assay was performed after treatment with or without 2 or 10 µM PTD-DBM. Representative images (left) and migrating cell numbers (right) are shown (**, P < 0.005; ***, P < 0.0005; n = 3 independent experiments). The widths of the wounds scratched at the start of the assay are indicated with dashed lines. (I) ICC staining for phalloidin (left) and α-SMA (right) was performed in human dermal fibroblasts treated with or without 2 or 10 µM PTD-DBM. Representative ICC images are shown (n = 3 independent experiments). Bars: (C and I) 50 µm; (H) 500 µm. Means ± SD.