1. Volume 50, Issue 3, Pages 479-488 (March 2009)
Targeted delivery of siRNA against hepatitis C virus by apolipoprotein A-I-bound cationic liposomes 
Soo In Kim, Duckhyang Shin, Hyeon Lee, Byung-Yoon Ahn, Yeup Yoon, Meehyein Kim 
Journal of Hepatology 
Volume 50, Issue 3, Pages (March 2009)
DOI: /j.jhep
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

2. Fig. 1
Inhibitory effect of HCV core-specific siRNA in Huh7 cells in vitro. (a) Schematic representation of pCEP4-HA-CE1E2 plasmid expressing viral core, E1, and E2 proteins of genotype 1b under the HCR and AAT promoter, and the location of restriction enzyme sites. HCR, hepatic control region; PAAT, alpha1-antitrypsin promoter. The nucleotide sequence represents the target sequence for HCV core-specific siRNA, siHCc. Its nucleotide position within the full length HCV genome is numbered. (b) Western blot analysis of pCEP4-HA-CE1E2-transfected Huh7 cell total lysates on day 2 using antibodies against HCV core, E2 and cellular SR-BI proteins. β-Actin was used as an internal loading control. (c) Western blot analysis showing cytosolic (C) and membrane (M) SR-BI levels in mock and pCEP4-HA-CE1E2-transfected Huh7 cells. Na–K ATPase was used as a membrane marker. (d) Sequence of siHCC and relative serum stability of unmodified siHCc and 2′-OMe-modified siHCc-U. (e) Viral protein (core and E2) level detected by Western blot and (f) viral RNA level detected by Northern blot 2 days after transfection of pCEP4-HA-CE1E2 (target) only or with each indicated siRNA (siCont, siHCc, siHCc-U or siHBx-U) using Lipofectamine Both siCont and siHBx-U were used as negative controls.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

3. Fig. 2
Determination of formulation efficiency of DTC-Apo particles and serum stability of siRNA complexed with liposomes. (a) The self-assembled mixture of rhodamine-labeled DTC and apo A-I was loaded onto a gel filtration column, and both fluorescence intensity and protein concentration were measured in each fraction. Filled circle indicates protein concentration and empty circle means rhodamine intensity from DTC liposome. (b) Detection of apo A-I from representative fractions using apo A-I-specific monoclonal antibodies by Western blot. (c) Naked siHCc, or liposome (DTC or DTC-Apo)-complexed siHCc was incubated at 37°C in 50% human serum. Samples were harvested at the indicated time points and analyzed by 15% non-denaturing SDS–PAGE.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

4. Fig. 3
Liver-targeted delivery and cellular uptake of fluorescence-labeled oligomer delivered by DTC-Apo in mice. (a) FITC-dsDNA (green) was complexed with DTC or DTC-Apo, and then intravenously injected into mice. Liver, kidney, lung, and spleen were applied to fluorescence microscopic analysis at 1h after injection (upper). Hematoxylin–eosin staining (lower). Original magnification=400×. (b) Cy3-labeled dsDNA (red) was complexed with DTC or DTC-Apo, and then intravenously injected into mice. At 24h, liver sections were harvested and then counterstained with DAPI (blue) and Oregon Green 488 phalloidin (green) to visualize cell nuclei and membranes, respectively. Images were analyzed using fluorescence microscopy. Original magnification=400×.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

5. Fig. 4
Inhibition of HCV gene expression by DTC/siHCc and DTC-Apo/siHCc in mouse liver. (a) Detection of HCV core and cellular SR-BI protein expression in liver of normal and pCEP4-HA-CE1E2-treated mice using Western blot analysis (n=3). (b) Dose-dependent reduction of core protein expression by DTC-Apo/siHCc in vivo. Relative hepatic core protein expression levels were quantified on day 2 after intravenous administration of DTC-Apo/siCont (2mg/kg), or siHCc complexed with either DTC or DTC-Apo at an siRNA dose of 0.25, 0.5, 1 or 2mg/kg (n=4). ∗P<0.05 and ∗∗P< versus DTC-Apo/siCont-treated group.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

6. Fig. 5
Duration of in vivo anti-viral activity of unmodified and chemically-modified HCV-specific siRNA. (a) Gene silencing was examined after intravenous administration of siHCc and siHCc-U, complexed with DTC-Apo, using Western blot analysis with mouse liver tissues (expressing HCV structural proteins) on days 2, 4, and 6. Non-immunostimulatory siHBx-U was used as a negative control (n=3). For the target only group, n=2. (b) The ratio of HCV core and β-actin protein from the Western blots (a) was calculated and expressed as a group average relative to the irrelevant siRNA-treated group (siHBx-U). ∗P<0.05 and ∗∗P<0.01 versus the DTC-Apo/siHBx-U group.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

7. Fig. 6
Relative effect of DTC/siLuc and DTC-Apo/siLuc on luciferase gene expression in normal and SR-BI−/− knockout mice. (a) Western blot analysis for measuring hepatic SR-BI expression in wild-type (SR-BI+/+, mouse no. 1–3) and knockout mice (SR-BI−/−, mouse no. 4–6). β-Actin was used as an internal loading control. (b) All mice were hydrodynamically injected with the luciferase expression DNA and then 24h later treated intravenously either with DTC/siLuc (no. 1 and 4) or DTC-Apo/siLuc (no. 2, 3, 5, and 6) at an siRNA dose of 1mg/kg per mouse. Whole body images were obtained on day 1 after siRNA treatment.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions

8. Fig. 7
Tissue microarray analysis of SR-BI expression on human liver tissues. (a) Six human liver sections of HCV-derived hepatocellular carcinoma were stained with an anti-SR-BI antibody and its secondary antibody-HRP-conjugated polymer, and then visualized with an immunoperoxidase substrate. (b) Human hepatocellular carcinoma tissues with HBV infection and cholangiocarcinoma samples were used as positive and negative control, respectively. Original magnification=400×.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2008 European Association for the Study of the Liver Terms and Conditions