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hep22465-SupplementaryFigure1.tif6592KSupplementary Figure 1. Analysis of polarity and TJ-associated proteins in Huh7, HCV-G1 and Caco-2 cells. (A) Huh7 (left), HCV-G1 (center) or Caco-2 (right) cells were grown on Transwell filters for 72h (Huh7 and HCV-G1 cells) or 10 days (Caco-2 cells) and processed for immunofluorescence with anti-Na-K ATPase (lateral marker [34], green) and ZO-1 (red) Abs, being shown the x-z sections. Z-sections were compiled by taking 0.6 μm steps through each x-y section. (B) Localization of occludin (green) and ZO-1 (red) in Transwell-cultured Huh7 (left), HCV-G1 (center) and Caco-2 (right) cells was analyzed by immunofluorescence (top, x-z sections; bottom, merged projection of confocal stacks). (C) Paracellular permeability of control or calcium-depleted Huh7, HCV-G1 and Caco-2 cells was determined as described in Supp. M&M and Fig. 1 legend. Results are expressed as the mean value ± SD of six experiments.
hep22465-SupplementaryFigure2.tif3498KSupplementary Figure 2. Characterization of genomic and subgenomic HCV replicon-containing clones. (A) Top: Western blot analysis of the HCV proteins core, E2 and NS5A expression in Huh7 cells and clones harbouring the genomic replicon I 389 /Core-3'/5.1 (HCV-G1, HCV-G5) or the subgenomic replicon I 377 /NS3-3' (HCV-NS18, HCV-NSA). After 48 hours of culture, cell lysates were analyzed for core, E2 and NS5A expression by Western blot. Analysis of p53 protein levels determined relative equal amounts of protein in all lanes. Molecular weight markers are indicated on the right (in kilodaltons). Bottom: HCV RNA levels. Samples of 1 μg RNA were analyzed by real-time RT-PCR using specific primers to determine HCV RNA levels. Histone H3 mRNA levels were used for sample normalization. Data are expressed as HCV RNA copies/μg total RNA. Data are represented as the mean value ± SD of four experiments. (B) Immunofluorescence analysis of core, E2 and NS5A expression in Huh7 (a-c), genomic [HCV-G1 (d-f), HCV-G5 (g-i)] and subgenomic clones [HCV-NS18 (j-l), HCV-NSA (m-o)]. Cells were fixed and processed for immunostaining with anti-core (a, d, g, j, m), anti-E2 (b, e, h, k, n) and anti-NS5A (c, f, i, l, o) specific Abs. Cells were stained with 4',6-diamidino-2-phenylindole (DAPI) to localize nuclei. Bar, 50 μm. Results are representative of five separate experiments.
hep22465-SupplementaryFigure3.tif4902KSupplementary Figure 3. Immunofluorescence analysis of TJ-associated proteins in additional replicon clones. Occludin and ZO-1 localization in the rest of genomic (top) and subgenomic (non-structural, bottom) HCV isolated clones after 48 hours of cell culture. Results are representative of five separate experiments.
hep22465-SupplementaryFigure4.tif3452KSupplementary Figure 4. Effect of dominant negative constructs inhibiting clathrin and clathrin/caveolin endocytic pathways on HCV-G1 cells. Cells were transiently transfected with plasmids harbouring dominant negative constructs inhibiting clathrin (top, GFP-EPS15DIII) or clathrin/caveolin endocytic pathways (bottom, Dyn2K44A-HA). After 48 hours of culture, cells were incubated with Texas Red-labelled transferrin (see Supp. M&M) and either mounted (for GFP-EPS15DIII transfectants) or processed for immunofluorescence with anti-HA and Alexa 488-conjugated goat anti-mouse (for Dyn2K44A-HA transfectants). Transferrin internalization (red) was severely impaired in transfected cells (green).
hep22465-SupplementaryFigure5.tif3408KSupplementary Figure 5. Analysis of occludin localization in Huh7 cells co-cultured with genomic replicon-containing clones. After transfection of Huh7 cells with pEGFP-C1, GFP-expressing cells were sorted, mixed with non-transfected Huh7 (top) or HCV-G1 (bottom) cells and seeded onto glass coverslips. After 48 hours of culture, localization of occludin was analyzed by immunofluorescence using anti-occludin (red) specific Ab. Bar, 20 μm. Arrow: the intercellular occludin staining between Huh7 cells (expressing GFP) is not altered when co-cultured with HCV-G1 cells.
hep22465-SupplementaryFigure6.tif5159KSupplementary Figure 6. Effect of HCV structural proteins on the localization of Huh7 TJ-associated proteins. Parental Huh7 cells were transfected with plasmids coding for HCV structural proteins (+ STR) or core protein (+ Core) and the distribution of occludin and viral proteins was analyzed by confocal immunofluorescence. Transfected Huh7 cells were grown on coverslips for 48 hours, fixed and processed for double-label immunofluorescence using anti-occludin (green) and anti-core or anti-E2 (red) Abs. Representative confocal images of cells are shown.
hep22465-Supplementarymaterialsandmethods.doc51KSupplementary materials and methods

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