Figure 1.
Figure 1. Generation of hepatocyte-specific Smad7-overexpressing mice and liver status. (A) Schematic presentation of the Smad7-expression cassette: downstream of the albumin regulatory elements (Alb.Enh.-Promotor) are globin intron sequences (Intron) and a lac-Z reporter gene (β-Gal) with polyA signal (pA), flanked by 2 loxP sites (indicated by blue triangles). Downstream of the second loxP site is the murine Smad7 complementary DNA with another polyA signal. The construct is flanked by insulator sequences (Ins.) to minimize the influence of neighboring genomic regulatory sequences. (B) LacZ staining of the livers of wild-type (wt) mice and Smad7-tg single-transgenic animals showing a clear and strong signal; liver sections from 2 Alb-Smad7–transgenic animals are shown to demonstrate partial to no staining reactions (original magnification ×40). (C-D) Presence of hepatic fibrosis was evaluated by immunohistochemistry for α-smooth-muscle actin (α-SMA) expression, extracellular matrix–producing cells (scale bar, 50 μm; inset scale bar, 20 μm), and Sirius red (scale bar, 50 μm; inset scale bar, 20 μm), respectively. (E) Relative messenger RNA (mRNA) expression of Smad7 in the liver of Alb-Smad7-tg (n = 7) and control mice (n = 5), measured by quantitative real-time polymerase chain reaction (PCR). (F) Immunohistochemistry for Smad7 expression (scale bar, 50 μm) on liver sections from control and Alb-Smad7-tg mice (arrows indicate positive brown-colored staining). (G-H) Immunoblot analysis and relative quantification of pSmad1, total Smad1, and β-actin proteins in the livers of Alb-Smad7-tg and control mice (n = 3 per group). (I-J) Immunohistochemistry for Smad2 phosphorylation and quantification of pSmad2+ hepatocyte nuclei per field on liver sections from Alb-Smad7-tg and control mice (I: scale bar, 200 μm; inset scale bar, 100 μm). Images are representative staining of 2 to 3 mice per group. Data were analyzed using GraphPad Prism software and results are shown as mean ± standard error of mean (SEM). For statistical analysis, a nonparametric distribution and the Mann-Whitney U test were used. *P < .05; **P < .005; ***P < .0005.

Generation of hepatocyte-specific Smad7-overexpressing mice and liver status. (A) Schematic presentation of the Smad7-expression cassette: downstream of the albumin regulatory elements (Alb.Enh.-Promotor) are globin intron sequences (Intron) and a lac-Z reporter gene (β-Gal) with polyA signal (pA), flanked by 2 loxP sites (indicated by blue triangles). Downstream of the second loxP site is the murine Smad7 complementary DNA with another polyA signal. The construct is flanked by insulator sequences (Ins.) to minimize the influence of neighboring genomic regulatory sequences. (B) LacZ staining of the livers of wild-type (wt) mice and Smad7-tg single-transgenic animals showing a clear and strong signal; liver sections from 2 Alb-Smad7–transgenic animals are shown to demonstrate partial to no staining reactions (original magnification ×40). (C-D) Presence of hepatic fibrosis was evaluated by immunohistochemistry for α-smooth-muscle actin (α-SMA) expression, extracellular matrix–producing cells (scale bar, 50 μm; inset scale bar, 20 μm), and Sirius red (scale bar, 50 μm; inset scale bar, 20 μm), respectively. (E) Relative messenger RNA (mRNA) expression of Smad7 in the liver of Alb-Smad7-tg (n = 7) and control mice (n = 5), measured by quantitative real-time polymerase chain reaction (PCR). (F) Immunohistochemistry for Smad7 expression (scale bar, 50 μm) on liver sections from control and Alb-Smad7-tg mice (arrows indicate positive brown-colored staining). (G-H) Immunoblot analysis and relative quantification of pSmad1, total Smad1, and β-actin proteins in the livers of Alb-Smad7-tg and control mice (n = 3 per group). (I-J) Immunohistochemistry for Smad2 phosphorylation and quantification of pSmad2+ hepatocyte nuclei per field on liver sections from Alb-Smad7-tg and control mice (I: scale bar, 200 μm; inset scale bar, 100 μm). Images are representative staining of 2 to 3 mice per group. Data were analyzed using GraphPad Prism software and results are shown as mean ± standard error of mean (SEM). For statistical analysis, a nonparametric distribution and the Mann-Whitney U test were used. *P < .05; **P < .005; ***P < .0005.

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