PMC

Observation of the liver. Livers were obtained from mice treated with Ringer's solution + pPK9a + ZnSO₄ for 24 h (A), Ringer's solution + pPK9a + ZnSO₄ for 48 h (B), Ringer's solution + pPK9a for 48 h (C), and vehicle (injection free) for 48 h (D).

Observation of liver fibrosis in transgenic mice. (A) Serum ALT levels. (B) Hydroxyproline content in liver. (C) Col 1A2 mRNA levels measured by real-time quantitative PCR. All samples were collected 48 h after gene transfer and induction with ZnSO₄. Values are represented as mean ± SD. *, p < 0.05; **, p < 0.001; compared with other groups; unpaired t-tests.

Enhancement of Sp1 binding activity by gene transfer accompanying with ZnSO₄ treatment. (A) Measurement of Sp1 binding activity performed by EMSA. Nuclear proteins were extracted from the livers of pPK9a-transferred mice administrating ZnSO₄-contained water for 1–5 days. Two μg of nuclear extract was subjected to ³²P-labelled probe and the Sp1-DNA complex was analyzed on a 4% native polyacrylamide gel. (B) Confirmation of Sp1 and Sp3 binding by supershift assay. Sp1 and Sp3 antibodies were added to the reaction mixtures for supershift assays. The shifted and supershifted bands were indicated by arrows.

Upregulation of α-SMA and ECM by gene transfer and ZnSO4 treatment in liver. (A) Detection of α-SMA by immunohistochemistry. Forty eight h after hydrodynamics-based injection of pPK9a, the mice were sacrificed and the liver sections were subjected to immunostaining. Dark brown granules represent α-SMA signals stained by α-SMA-specific antibody and indicated by arrows. (B) Detection of ECM and collagen by Masson's trichrome staining. The cytoplasm was stained red and collagen fibers in ECM were blue-green. The collagen signals were indicated by arrows. Representative liver sections of α-SMA and collagen from experimental I-IV groups: (I) Ringer's solution + pPK9a + ZnSO₄ 48 h. (II) Ringer's solution + ZnSO₄. (III) Ringer's solution + pPK9a. (IV) Mice without hydrodynamics-based injection. Bar = 0.2 mm.

Conditional regulation of TGF-β1 expression in mice. (A) Serum TGF-β1 levels in hydrodynamics-based gene transferred mice. Values are represented as mean ± SD. *, p < 0.01; compared with pPK9a alone (48 h); #, p < 0.05; compared with pPK9a + ZnSO₄ (24 h); and, p < 0.05; compared with pPK9a + ZnSO₄ (48 h); unpaired t-tests. (B) RT-PCR analysis for TGF-β1 mRNA expression at 48th h in the liver of gene transferred mice after injection with pPK9a. (C) Protein expression of TGF-β1 and α-SMA at 48th h in gene transferred mice after injection with pPK9a. (D) Photomicrographs of immunohistochemical analysis (upper panel) and bright field (lower panel) for TGF-β1 expression at 48 h in liver sections: I. Ringer's solution + pPK9a and ZnSO₄free. II. Ringer's solution only. III. Ringer's solution + pPK9a + ZnSO₄. Bar = 0.2 mm. (E) TGF-β1 protein expression between 1 to 5 days.