CCN5 prevents TGF-β-induced fibroblast-like phenotypic transformation of ARPE-19 cells.

(A) ARPE-19 cells were grown until 100% confluent and cultured in the presence of TGF-β and AdLacZ or AdCCN5. (B) Bright field microscopy images showing the change in the morphology of ARPE-19 cells from a monolayer of cuboidal cells to mesenchymal-like cells upon treatment with 10 ng/ml TGF-β. This change was not observed in cells pretreated with AdCCN5. Original magnification: 100×. Scale bar: 200 μm. (C) Total cell lysates obtained under the same conditions as 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 (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. Areas of collagen gel contraction were measured after 48 hours. Representative images of collagen gel lattices are shown (left), and areas of gel contraction were quantified (right) (n = 5). Error bars = S.D. *p < 0.05, **p < 0.01.