Stability of ABCB4-wt and ABCB4-ΔQNL.

<p>(A) Stability of ABCB4-wt or ABCB4-ΔQNL was analyzed in stably transfected HepG2, after inhibiting protein synthesis with cycloheximide (25 μg/mL). ABCB4 was detected at the indicated time points in cell lysates by immunoblotting, using equal amounts of total proteins per lane. (B) Amounts of ABCB4 were quantified from chase experiments. The amount of ABCB4 at time zero was considered as 100%. Remaining ABCB4 at later time points was expressed as percentage of time zero. Means (± SEM) of three independent experiments are shown. <i>* P</i><b><</b>0.05 at all points.</p

Effect of overexpression of the PDZ domains of EBP50.

<p>(A) ABCB4-wt- or ABCB4-ΔQNL-expressing HepG2 cells were transiently transfected with the Flag-tagged EBP50 PDZ domains (PDZ1+PDZ2-Flag) or with the empty vector-Flag (control vector). ABCB4 was detected by immunoblotting from cell lysates. (B) Amounts of ABCB4 were quantified from immunoblots by densitometry. ABCB4 levels were expressed as a percentage of total expression in HepG2 cell transfected with control vector. (C) ABCB4-wt- or ABCB4-ΔQNL-expressing HepG2 cells were transiently transfected with the Flag-tagged EBP50 PDZ domains (PDZ1+PDZ2-Flag). Cells were fixed, permeabilized and stained with the anti-Flag antibody followed by anti-ABCB4 antibody and then incubated with Alexa-Fluor-594-and 488-conjugated secondary antibodies and visualized by confocal microscopy. Representative immunofluorescence images are shown. Asterisks indicate bile canaliculi. Bars, 10 μm. (D) The amount of ABCB4 at the bile canaliculi was quantified in HepG2 transfected cells (PDZ1+PDZ2-Flag(+)) and compared to that observed in control adjacent non-transfected cells (PDZ1+PDZ2-Flag(-)). Means (± SEM) of at least three independent experiments are shown. *<i>P</i><0.01 for ABCB4-wt; n.s., not significant; a.u., arbitrary units.</p

Expression and localization of ABCB4-wt and ABCB4-ΔQNL.

<p>(A) ABCB4 was detected by immunoblotting from cell lysates of HepG2 cells stably expressing ABCB4-wt or ABCB4-ΔQNL. (B) HepG2 cells stably expressing ABCB4-wt or ABCB4-ΔQNL were fixed with methanol/acetone, processed for immunofluorescence using the monoclonal P3II-26 antibody and Alexa 488-conjugated anti-mouse IgG and visualized by confocal microscopy. Nuclei were stained with DRAQ 5 (blue). Asterisks indicate bile canaliculi. Cell contours are indicated by dotted lines. Bars, 10 μm. Representative of three experiments.</p

Effect of EBP50 silencing.

<p>(A) HepG2 cells expressing ABCB4-wt or ABCB4-ΔQNL were transfected with EBP50 siRNA or scramble siRNA (Scr). After 60 hours of transfection, cell lysates were subjected to western blot analysis. (B) Amounts of ABCB4 were quantified from immunoblots by densitometry. ABCB4 levels were expressed as a percentage of total expression in HepG2 cell transfected with scramble siRNA. Means (±SEM) of at least three independent experiments are shown. *<i>P</i><0.05 for ABCB4-wt; n.s., not significant.</p

The internalization of ABCB4-ΔQNL is accelerated.

<p>(A) Transiently transfected HEK 293 cells were incubated for 30 minutes at 0°C with anti-myc antibody. After surface labeling, cells were incubated at 37°C for 60 minutes. At the end of the 37°C incubation, non-internalized antibody-antigen complexes were removed by acid washing. Cells were fixed, permeabilized and internalized antibodies were visualized with Alexa-Fluor 488-conjugated secondary antibodies and examined by confocal microscopy. Representative immunofluorescence images are shown. Nuclei were stained with DRAQ 5 (blue). Bars, 10 μm. (B) The amount of internalized ABCB4 was measured in cells transfected with 3xmyc-ABCB4-wt or 3xmyc-ABCB4-ΔQNL using ImageJ 1.41 software. Means (± SEM) of at least three independent experiments are shown. *<i>P</i><0.001. a.u., arbitrary units.</p

The C-terminus of ABCB4 ends by a PDZ-like motif.

<p>(A) Schematic representation of ABCB4. ABCB4 contains two transmembrane domains (TMD1 and TMD2) and two nucleotide binding domains (NBD1 and NBD2). The two glycosylation sites in the first extracellular loop are indicated. The amino acid sequence of the intracytoplasmic C-terminal domain of the human ABCB4 isoform A (NP_000434.1) and the human ABCB1 (NP_000918.2) are shown. Green letters indicate the PDZ-like motif of ABCB4. (B) A sequence alignment of known PDZ motifs found in ABC family members is shown. ABCC2, ABCC4 and ABCC7 display class I PDZ consensus motifs.</p

ABCB4 colocalizes and coimmunoprecipitates with EBP50.

<p>(A) ABCB4-wt-expressing HepG2 cells were fixed, permeabilized and stained with anti-ABCB4 antibody followed by anti-EBP50 antibody and then incubated with Alexa-Fluor-488-and 594-conjugated secondary antibodies and visualized by confocal microscopy. Nuclei were stained with DRAQ 5 (blue). Asterisks indicate bile canaliculi. Bar, 10 μm. (B) Cell lysates of HepG2 cells stably transfected with ABCB4-wt or ABCB4-ΔQNL or cell lysates of primary human hepatocytes (PhH) were incubated with anti-ABCB4 antibody or mouse imununoglobulin G (IgG) covalently linked to agarose beads. The coimmunoprecipitated complex was immunoblotted with anti-ABCB4 and anti-EBP50 antibodies.</p

Infection status of HLMF inoculated with HCVcc.

<p>HLMF were inoculated with JFH1-HCVcc 24 hours after plating. Parameters of HCV infection were monitored at the indicated days after inoculation, and in non-infected HLMF or 3T3 and 293T cells inoculated with JFH1-HCVcc, as controls. Strand-specific HCV RNA was measured by RT-PCR: <b>(A)</b> In lysed cells, <b>(B)</b> In filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA per μg of total cellular RNA or per ml of supernatant (means ± SD, Ten cell preparations). <b>(C)</b> Intracellular levels of HCV NS3 and core proteins were analyzed by Western blot after 72h post infection in comparison with Huh7.5 or 3T3 cells inoculated with JFH1-HCVcc and with non-infected cells. <b>(D)</b> Levels of HCV core protein in filtered culture supernatants were measured by chemiluminescent microparticle immunoassay (means ± SD, two cell preparations). The blots and histograms are representative of seven HLMF preparations. <b>(E)</b> Immunofluorescence on HLMF with co-staining (CD90 and HCV core protein) and on Huh7.5 with co-staining (CLDN-1 and HCV core protein). <b>(F)</b> Inhibition of HCVcc infection by anti-CD81 antibody or IFN-lpha in HLMF. HLMF were incubated with (a-c) an anti-CD81 neutralizing monoclonal antibody or an isotype-matched control antibody, added 1 h before HCVcc inoculation, or with (d-f) IFN-lpha or a vehicle after HCVcc inoculation. The concentrations tested are indicated. HCV infection was evaluated three days after the inoculation by RT-PCR analysis of strand-specific HCV RNA (a, b, d, e) in lysed cells, and (c and f) in filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA per μg of total cellular RNA or per ml of supernatant, from triplicate independent experiments.</p

Expression of HCV entry receptors in human liver myofibroblasts (HLMF).

<p>HLMF were subjected to the analysis of HCV entry receptors (CD81, LDLR, CLDN-1, OCLN and SR-BI) using four techniques. <b>(A)</b> Quantitative RT–PCR. Histograms represent mRNA levels relative to Huh7.5 in PHH, HLMF, HepG2 and the T98G cell line (means ± SD from 7 cell preparations). <b>(B)</b> Flow cytometry (CD81 and LDLR). The histograms of mean fluorescence showed that Huh7.5, PHH and HLMF expressed CD81 and LDLR, whereas HepG2 expressed LDLR but not CD81. Appropriate IgG controls were used as negative controls. <b>(C)</b> Immunofluorescence. HLMF, Huh7.5, PHH, HepG2 and the T98G line were stained for HCV receptors and imaged with a Leica DMR inverted microscope using LAS image analysis (Original magnification X40). The Flow cytometry and IF results are representative of seven HLMF preparations. <b>(D)</b> CLDN-1, OCLN and SR-BI detection using Western blot analysis. Huh7.5 and PHH were used as positive controls and T98G as a negative control for all receptors. The blots are representative of three HLMF preparations.</p

Infection status of HLMF from HCV-infected patients.

<p>HLMF were prepared from HCV-infected subjects and monitored after 7 days of primary culture (P0) and after 1 or 2 passages (P1, P2): <b>(A)</b> to ascertain the absence of hepatocyte contamination using RT-PCR analysis of HNF-1ß, CYP2E1 and albumin. The results are expressed as mRNA levels in individual HLMF preparations relative to those in primary cultures of human hepatocytes (PHH); to detect HCV infection using RT-PCR analysis of strand-specific HCV RNA <b>(B)</b> in lysed cells and <b>(C)</b> in filtered culture supernatants. Histograms represent the copies of strand-specific HCV RNA perμg of total cellular RNA or per ml of supernatant, from two cell preparations.</p