Volume 129, Issue 5, Pages 1654-1662 (November 2005) Short Hairpin RNA Modulates Transforming Growth Factor β Signaling in Life- Threatening Liver Failure in Mice  Yoshiaki Mizuguchi, Shigeki Yokomuro, Takuya Mishima, Yasuo Arima, Tetsuya Shimizu, Yutaka Kawahigashi, Tomohiro Kanda, Hiroshi Yoshida, Toshihiro Takizawa, Takashi Tajiri  Gastroenterology  Volume 129, Issue 5, Pages 1654-1662 (November 2005) DOI: 10.1053/j.gastro.2005.08.013 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Silencing of mouse TGF-βRII by shTGF-βRII in BNL CL.2 cells. Cells were transfected 3 times with shNS, shGFP, shTGF-βRII-1, or shTGF-βRII-2 (n = 6 per group). Values are given as means ± SD. **P < .01; ***P < .001 compared with shNS (solid bar). (A) Predicted structures of the coding sequence for shRNAs targeting mouse TGF-βRII with secondary structure prediction. (B and C) Real-time PCR analysis of the silencing effects on TGF-βR2/GAPD (B) and TGF-βR1/GAPD (C) mRNA expression. (D) Immunofluorescence staining assessment of the silencing effects on TGF-βRII protein expression. Representative double staining with TGF-βRII-specific antibody (top), DAPI staining of DNA (middle), and overlaid images with TGF-βRII-specific antibody and DAPI (bottom; scale bar indicates 50 μm). (E) Representative double fluorescence staining (TGF-βRI + DAPI) of cells (scale bar indicates 50 μm). (F) Western blotting assessment of the silencing effects on TGF-βRII protein expression. Blotting was performed with TGF-βRII- or GAPDH-specific antibodies. (G and H) Real-time PCR analysis of the effect of shTGF-βRII treatment on mRNA expression by IFN-responsive genes (PRKR/GAPD and OAS1A/GAPD). Gastroenterology 2005 129, 1654-1662DOI: (10.1053/j.gastro.2005.08.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Silencing human TGF-βRII with shTGF-βRII in HuCC-T1 cells. Cells were transfected 3 times with shNS, shGFP, or shTGF-βRII-A (n = 5 or 6). Values are expressed as means ± SD. ***P < .001 compared with shNS (solid bar). (A) Predicted structures of the coding sequence for shRNAs targeting human TGF-βRII with secondary structure prediction. (B) Real-time PCR analysis of the silencing effects on TGF-βR2/GAPD mRNA expression. (C) Immunofluorescence staining assessment of the silencing effects on TGF-βRII protein expression. Representative double staining with TGF-βRII-specific antibody (top), DAPI staining of DNA (middle), overlaid images with TGF-βRII-specific antibody, and DAPI (bottom; scale bar indicates 50 μm). (D) Western blotting assessment of the silencing effects on TGF-βRII protein expression. Blotting was performed with TGF-βRII or GAPDH specific antibodies. Gastroenterology 2005 129, 1654-1662DOI: (10.1053/j.gastro.2005.08.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 In vitro suppression of TGF-β signaling by shTGF-βRII protects cells from injury. Cells were treated with (A, B, D–G, 2500 pg/mL; C, 2.5, 25, 250, 2500 pg/mL) or without rhTGF-β1 after being transfected 3 times with shNS, shGFP, shTGF-βRII-1, or shTGF-βRII-2 (n = 5 or 6). Values are expressed as means ± SD. *P < .05; **P < .01; ***P < .001 compared with shNS (solid bar). (A and B) Western blotting assessment of the silencing effects on suppression of SMAD2 activation 30 minutes, 3 hours (A), and 48 hours (B) after rhTGF-β1 treatment. Blotting was performed with specific antibody for phosphorylated-SMAD2 (PSMAD2), SMAD2, or GAPDH. (C and D) Real-time PCR assessment of the silencing effects on suppression of TGF-β-responsive gene expression. Results are expressed as gene/GAPD. NS denotes shNS; 1 denotes shTGF-βRII-1. (E) BrdU incorporation assay of the silencing effects on the maintenance of cell proliferation (double staining with BrdU-specific antibody and DAPI). Scale bar indicates 100 μm. (F) TUNEL assay of the silencing effects on suppression of apoptosis. Cells counterstained with DAPI. Scale bar indicates 100 μm. (G) Morphologic assessment of the silencing effect on cell viability with H&E staining. Scale bar indicates 400 μm. Gastroenterology 2005 129, 1654-1662DOI: (10.1053/j.gastro.2005.08.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 In vivo suppression of TGF-β signaling by shTGF-βRII protects the liver from injury. Mice were treated intravenously with a sublethal dose of Fas agonistic Jo-2 antibody (0.10–0.15 μg/g) with shNS, shTGF-βRII-1, or shTGF-βRII-2 (n = 4–6). The control experiment with vehicle-treated mice was performed using an equal volume of PBS, followed by no treatment with the hepatotoxic reagent. Values are expressed as means ± SD. *P < .05; **P < .01; ***P < .001 compared with shNS (solid bar). (A) Confirmation of efficient and specific delivery of the shRNA-expressing plasmid into mice hepatocytes. (a) Representative confocal microscopic images of mouse liver after a single hydrodynamic injection of shNS (100 μg) and EGFP expression plasmid (p-EGFP-C1, 100 μg). Scale bar indicates 400 μm. (b–d) Confirmation of specific delivery. Representative immunofluorescence microscopic images of cells expressing EGFP using DAPI staining (b), immunofluorescent staining using albumin-specific antibody with DAPI (c), and overlaid images (d). Note that the image overlay demonstrates the colocalization of EGFP and albumin, which is a specific marker of hepatocytes. The arrows indicate a cell expressing EFGP, the arrowheads indicate a nonparenchymal cell, and S indicates a sinusoid. Scale bar indicates 50 μm. (B) Real-time PCR assessment of the silencing of TGF-βR2 and its effect on suppression of TGF-β-responsive gene expression and on activation of IFN-responsive gene expression. Results are expressed as gene/GAPD. (C) BrdU incorporation assay of the silencing effects on the maintenance of cell proliferation (double staining with BrdU-specific antibody and DAPI). Scale bar indicates 100 μm. (D) TUNEL assay of the silencing effects on the suppression of apoptosis. Cells counterstained with DAPI. Scale bar indicates 200 μm. (E) Morphologic assessment of the silencing effect on cell viability using H&E staining and of stellate cell activation using immunofluorescent staining for α-smooth muscle actin with DAPI. Note that, in all of the models, stellate cells have very little, if any, labeling for α-smooth muscle actin, whereas the smooth muscle layer in vessels is strongly labeled. Scale bar indicates 50 μm (high magnification) and 200 μm (low magnification), respectively. Gastroenterology 2005 129, 1654-1662DOI: (10.1053/j.gastro.2005.08.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Modulating TGF-β signaling in mice with ALF. (A and B) Therapeutic effects of silencing on the release of serum AST and ALT. Mice were treated intravenously with a sublethal dose of Jo-2 antibody (0.10–0.15 μg/g) (A) or treated intravenously with a 75% lethal dose of LPS (5–6 μg/g) (B) after shNS, shTGF-βRII-1, or shTGF-βRII-2 injection. The control experiment with vehicle-treated mice was performed using an equal volume of PBS, followed by no treatment with the hepatotoxic reagent. Values are expressed as mean ± SD (n = 4–6). ***P < .001 compared with shNS (solid bar). The bar for the vehicle-treated mice is not visible because the values were within the normal limits (AST, 20–48 U/L; ALT, 10–40 U/L). (C) The effect of LPS injection on the release of serum TNF-α. Values are expressed as mean ± SD (n = 4). The bar for the vehicle-treated mice is not visible because the values were within normal limits (<25 pg/mL). (D and E) Mice were treated intravenously with a 50% lethal dose of Jo-2 antibody (0.20–0.25 μg/g) (D) or treated intravenously with a 75% lethal dose of LPS (5–6 μg/g) (E) and were followed up for 14 days. **P < .01. Gastroenterology 2005 129, 1654-1662DOI: (10.1053/j.gastro.2005.08.013) Copyright © 2005 American Gastroenterological Association Terms and Conditions