Figure 4

ADP355 inhibits fibrotic responses in normal and SSc skin fibroblasts. (a–d). Confluent cultures of normal dermal fibroblasts were preincubated with ADP355, followed by TGF-β2 for 24 h. (a) Total RNA was subjected to real-time qPCR. Results are mean ± SD of triplicate determinations. *p < 0.05, **p < 0.01. (b) Western blot of whole cell lysates. Representative results. Fold change in Type I collagen (Cgn I) levels relative to control is shown at bottom. (c) Fibroblasts were transiently transfected with [SBE]₄-luc reporter plasmids, and cell lysates were assayed for their luciferase activities. Results are mean ± SD of triplicate determinations. **p < 0.001 (d) Scratch assays were performed and the widths determined after 48 h. Results are mean ± SEM of triplicate determinations at three randomly selected sites. (e) 3D human skin equivalents populated with normal fibroblasts were incubated in media with ADP355 and TGF-β2 for 6 days. RNA was isolated for qPCR. Results are mean ± SD of triplicate determinations *p < 0.05, **p < 0.001. (f) SSc skin fibroblasts (n = 5) incubated with ADP355 for 24 h were immunostained with antibodies to αSMA (green) or type I collagen (Cgn1, red), or stained with DAPI (blue). Left panels, representative immunofluorescence photomicrographs (original magnification × 400). Right panels, immunofluorescence intensity. Results are means ± SD from five randomly selected hpf.