1. Date of download: 6/23/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Effect of Smad7 Gene Overexpression on Transforming Growth Factor β–Induced Retinal Pigment Fibrosis in a Proliferative Vitreoretinopathy Mouse Model Arch Ophthalmol. 2007;125(5):647-654. doi:10.1001/archopht.125.5.647 Expression of Smad7 and α-smooth muscle actin (α-SMA) in ARPE-19 cells. A, Western blotting detects marked expression of Smad7 in cells coinfected with Cre-expressing adenovirus and LoxP–neomycin resistance gene–LoxP (LNL)–Smad7 adenovirus (Smad7-Ad group) in the presence and absence of exogenous transforming growth factor β2 (TGF-β2). Cells treated with Cre-Ad only do not express Smad7 protein. Smad7 overexpression does not alter the protein expression level of α-SMA. B, Dual immunostaining detection of Smad7 (fluorescein) and α-SMA (rhodamine) in cells treated with either Cre-Ad or Smad7-Ad that had been incubated with TGF-β2. The α-SMA is readily observed in many cells in the Cre-Ad group, whereas faint α-SMA is seen in cells that lack Smad7 protein expression. Immunofluorescence staining was with 4′-6-diamidino-2-phenylindole nuclear staining. Reference bar = 20 μm. Figure Legend:

2. Date of download: 6/23/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Effect of Smad7 Gene Overexpression on Transforming Growth Factor β–Induced Retinal Pigment Fibrosis in a Proliferative Vitreoretinopathy Mouse Model Arch Ophthalmol. 2007;125(5):647-654. doi:10.1001/archopht.125.5.647 Effects of adenoviral Smad7 overexpression on transforming growth factor β (TGF-β)/Smad signal in ARPE-19 cells. A and B, Western blotting further confirms that overexpression of Smad7 decreases the level of expression of phosphorylated Smad2 at each time point. Total Smad2 level is unchanged with Smad7 overexpression. C, Immunocytochemical localization of phosphorylated Smad3 in cells shows that phosphorylated Smad3 locates to the nuclei of cells in the Cre-Ad group 1 hour after TGF-β2 addition, whereas in the Smad7-Ad group such nuclear accumulation of phosphorylated Smad3 is suppressed. D, Phosphorylated Smad2 is faintly observed at 0.5 hour, and it markedly accumulates in the nuclei at 1 hour in the Cre-Ad group. The immunoreactivity then decreases and almost disappears at 6 hours. On the other hand, in the Smad7-Ad group, up-regulation of phospho-Smad2 seems suppressed, and many cells without nuclear immunoreactivity are observed even 1 hour afterTGF-β2 addition. Reference bars = 50 μm. Numbered values are given in hours. Figure Legend:

3. Date of download: 6/23/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Effect of Smad7 Gene Overexpression on Transforming Growth Factor β–Induced Retinal Pigment Fibrosis in a Proliferative Vitreoretinopathy Mouse Model Arch Ophthalmol. 2007;125(5):647-654. doi:10.1001/archopht.125.5.647 Transforming growth factor β2 (TGF-β2) up-regulates TGF-β1 and collagen type I and enhances α-smooth muscle actin–positive cytoskeleton in ARPE-19 cells, and these effects are counteracted by Smad7 overexpression. A, Real-time reverse transcriptase– polymerase chain reaction shows that TGF-β2 up-regulates messenger RNA (mRNA) of TGF-β1 and collagen type Iα2 chain. The level of up-regulation of these components is counteracted by Smad7 gene transfer. B, Enzyme-linked immunosorbent assay also shows that Smad7 gene transfer partially reverses the increase of collagen type I in culture medium of ARPE-19 cells induced by TGF-β2. *P<.05. †P<.01. Error bars represent SD; +, increased; and −, decrease. C, Immunohistochemical analysis detected collagen type I in the cytoplasm of TGF-β2–treated control (Cre-adenovirus–treated) ARPE-19 cells (a), but this immunoreactivity is abolished by Smad7 gene transfer (c). Fibronectin deposition in control cells is seen in the presence of exogenous TGF-β2 (b), whereas much less fibronectin is detected in cells with Smad7 overexpression (d). Immunofluorescence staining was with 4′-6- diamidino-2-phenylindole nuclear dye. Reference bars = 25 μm (a, b, and d) and 50 mm (c). Figure Legend:

4. Date of download: 6/23/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Effect of Smad7 Gene Overexpression on Transforming Growth Factor β–Induced Retinal Pigment Fibrosis in a Proliferative Vitreoretinopathy Mouse Model Arch Ophthalmol. 2007;125(5):647-654. doi:10.1001/archopht.125.5.647 Adenoviral gene transfer of Smad7 inhibits multilayerization of retinal pigment epithelial (RPE) cells and their expression of α- smooth muscle actin after retinal detachment. A, Histologic findings (hematoxylin-eosin; original magnification ×20). On day 5, RPE cells have already elongated and formed a cell multilayer (arrows) in control (Cre-adenovirus–treated) eyes (a). This structure is also observed on day 10 (c) and day 21 (e). On the other hand, in eyes with Smad7 overexpression, RPE remains in a monolayer after retinal detachment on day 5 (b), day 10 (d), and day 21 (f). B, Expression pattern of α-smooth muscle actin (arrows) in RPE cells. On day 5, RPE cell multilayers begin to express α-smooth muscle actin in control (Cre-adenovirus–treated) eyes (a). The expression level increases on day 10 (c) and day 21 (e). In eyes with Smad7 overexpression, RPE remains negative for α-smooth muscle actin on day 5 (b), day 10 (d), and day 21 (f). C, Collagen-type I deposition in fibrotic tissue formed on the RPE. On day 21, multilayered cells contain collagen type I (arrows) (a), whereas monolayer RPE lacks type I collagen expression in an eye treated with Smad7 gene transfer (b). Immunofluorescence staining was with 4′-6-diamidino-2-phenylindole nuclear dye. Reference bars = 50 μm. Figure Legend:

5. Date of download: 6/23/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Effect of Smad7 Gene Overexpression on Transforming Growth Factor β–Induced Retinal Pigment Fibrosis in a Proliferative Vitreoretinopathy Mouse Model Arch Ophthalmol. 2007;125(5):647-654. doi:10.1001/archopht.125.5.647 Expression patterns of Smads in the retinal pigment epithelium (RPE) after retinal detachment. Smad7 protein is barely detected in the RPE on day 5 in a control (Cre-adenoviral [Cre-Ad]–treated) eye (A), whereas it is readily seen in an eye with Smad7 gene transfer (B). In the control eye on day 5, phopshorylated Smad2 is observed in the nuclei of RPE cells (C) but not in such cells in an Smad7-treated eye (D). The expression pattern of phosphorylated Smad3 is similar to that of phosphorylated Smad2 (E [control eye] and F [Smad7-treated eye]). The expression patterns of these components on day 10 are similar to those observed on day 5 (G-L). On day 21, the expression level of Smad7 is similar between a control (M) and a treated (N) eye. Nevertheless, nuclear accumulation of phosphorylated Smad2 (O and P) and phosphorylated Smad3 (Q and R) is more marked in a control eye (O and Q) compared with an Smad7 gene transfer eye (P and R). Immunofluorescence staining was with 4′-6-diamidino-2-phenylindole nuclear dye. Reference bar = 50 μm. Figure Legend: