CCN5 reverses TGF-β-induced fibrotic deformation of ARPE-19 cells.

(A) Fully confluent ARPE-19 cells were serum-starved for 24 hours, treated with TGF-β, and then infected with AdLacZ or AdCCN5. (B) Bright field microscopy images showing the change in the morphology of ARPE-19 cells from an organized epithelial cobblestone-like monolayer to fibroblast-like cells upon treatment with TGF-β in the absence of AdCCN5. Original magnification: 100×. Scale bar: 200 μm. (C) Total cell lysates prepared under the same conditions as shown in A were immunoblotted with antibodies against CCN5, α-SMA, vimentin, fibronectin, ZO-1, and occludin. GAPDH was used as a loading control. Quantified protein levels were normalized against the loading control (left bottom). Error bars = S.D. *p < 0.05, **p < 0.01. (D) Immunofluorescence images of ARPE-19 cells stained for ZO-1 and α-SMA. Nuclei were counterstained with Hoechst. Images are representative of at least three independent experiments. Original magnification: 200×. Scale bar: 50 μm. (E) Collagen gel lattices were generated and cultured under the same conditions as shown in A. Representative images of collagen gel lattices are shown (left), and areas of gel contraction were quantified (right) (n = 3). Error bars = S.D. *p < 0.05, **p < 0.01.