PrP follows a transcytotic route at steady state.

<p><b>(A)</b> After 5 days of polarization MDCK cells were biotinylated on the basolateral or apical membrane. After surface biotinylation cells were allowed to secrete in serum-free media for 3 hours. Media was collected, streptavidin-precipitated, PNG^ase treated and then analyzed by western blot using SHA31 antibody. <b>(B)</b> 1/10 of cell lysate and 1/5 of media before the streptavidin-precipitation was deglycosylated and run on western blot. <b>(C)</b> Transcytosis quantification. The relative amount of PrP, biotinylated on the basolateral membrane and streptavidin-precipitated in the apical membrane was normalized to the amount of PrP biotinylated on the apical membrane and precipitated from the apical media. Quantification was done on 4 independent experiments.</p

PrP distribution changes upon cyst maturation.

<p>MDCK PrP wt cells were plated in 2% Matrigel^™, and then fixed at 1 day (1 dpp), 2 days (2 dpp), 3 days (3 dpp) and 4 days (4 dpp) post plating. Cysts were co-stained with SAF32 (red), SHA31 (green), phalloidin-Alexa-647 (white) and DAPI (blue). Scale bar 10 μm. Representative images are shown from a total of 3 experiments.</p

Model of PrP trafficking in MDCK cells.

<p>In fully polarized epithelial cells PrP is exported from the Golgi to both apical and basolateral surfaces (40/60) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157991#pone.0157991.ref029" target="_blank">29</a>].<b>(1)</b> A significant part of PrP sorted from TGN to the apical membrane undergoes cleavage in the vesicular compartment or at the apical cell surface. (2) PrP sorted from TGN to the basolateral membrane stays intact. <b>(3)</b> Basolateral PrP is transcytosed and a part of it is cleaved on the way to the apical membrane. <b>(4)</b> Soluble N-terminal fragment is released to the apical media; full-length PrP as well as C-terminal fragment are shed from the apical media.</p

PrP wt undergoes basolateral to apical transcytosis in 2D polarized MDCK.

<p><b>(A)</b>. After 5 days of polarization MDCK cells were incubated with SHA31 (green) and SAF32 (red) in the basal media for 2 hours on ice, then filters were washed and incubated for +4°C as a control (left column) or for 3h at 37°C (right column). After fixation cells were permeabilized and stained with secondary antibodies and DAPI (blue). <b>(B)</b>. Quantification of the apical vs basolateral antibody distribution for 3h of incubation at 4°C (left) or at 37°C (right). Note that after the 3h of incubation SAF32 is present in intracellular sub-apical vesicles contributing to the quantification. The experiment was repeated 5 times, and a total of 100 cells were included in the analysis. (*p ≤ 0.05; ***p ≤ 0.001 Paired t test).</p

PrP cleavage through establishment of polarity in 2D MDCK cells.

<p><b>(A)</b> MDCK PrP wt was plated in Transwell^™ filters, then lysed at 6 hours (0 days), 1, and 3 days post plating. Cell lysates were PNG^ase treated and analyzed by western blot, revealed with SAF32 (upper panel) or SHA31 (lower panel) antibodies. <b>(B)</b> Left: Quantification of C-ter/PrP FL ratio through time. Right: normalization (using Coomassie blue staining) of C-ter and PrP levels with time. 4 independent experiments were quantified. <b>(C)</b> MDCK PrP wt was plated on Transwell^™ filters for 5 days. At 1, 3 and 5 days post plating growth media was replaced by serum-free media for 3 h. This media was collected and secreted proteins were methanol precipitated from the apical or basolateral media. Precipitated media was subjected to PNG^ase treatment and western blotting with SHA31 antibody (left) and SAF32 antibody (right). Of note, we detect 2 different bands around 15 kDa in the apically secreted C-terminal fragment, while we detect a single band of C-ter in the basolateral media. <b>(D)</b> Quantification of PrP FL revealed with either SAF32 or SHA31 (left panel), C-ter revealed with SHA31 (middle panel) secretion and N-ter revealed with SAF32 (right panel). All densitometry quantifications were normalized to day 1. 3 independent experiments were quantified. (*p ≤ 0.05; **p ≤ 0.01, Mann-Whitney test).</p

Different localization of PrP in non-polarized and polarized MDCK cells in 2D and 3D, revealed by using different antibodies.

<p><b>(A)</b> Schematic representation of mouse PrP α-cleavage site and the recognition sites of antibodies used in this paper. The C-terminal part of PrP (orange) is membrane-attached via GPI-anchor; it has 2 independently occupied glycosylation sites (orange circles). Proteolytic α-cleavage occurs at the position 109*110. After the cleavage C-terminal fragment (C-ter) stays on the plasma membrane. C-ter as well as PrP full-length (FL) is recognized by SHA31 antibody (orange). The N-terminal part (blue), becomes a soluble N-terminal fragment (N-ter) upon cleavage. N-ter and PrP FL are recognized by SAF32 antibody (blue). <b>(B), (C)</b> and <b>(D)</b> Immunofluorescence images of MDCK cells stably expressing PrP (MDCK PrPwt cells). Non-polarized cells—plated for 24 hours on Matek (B), polarized MDCK -5 days on Transwell filters (C) 3D polarized cyst- 5 days in Matrigel^™ (D) were fixed and immunostained for PrP using SAF32 antibody (red) and SHA31 antibody (green) and nuclei are stained with DAPI (blue). Scale bars 10 μm. In <b>(C)</b> serial confocal sections of 0,3 μm were collected from the top to the bottom of the cell monolayer. <b>(E)</b> Pearson’s R values revealing colocalization of SAF32 and SHA31 signal in non-polarized, 2D and 3D polarized states. (**p ≤ 0.01, Mann-Whitney test) <b>(F)</b> Quantification of apical vs. basolateral distribution of SAF32 and SHA31 in 2D polarized MDCK cells (left panel) and in 3D polarized MDCK cysts (right panel). These experiments were performed 3 independent times (20 cells quantified per experiment) and a total of 60 cells were used for the final quantification. (** p ≤ 0.01, paired t-test).</p

GW3965 treatment up-regulates ABCA1 expression and its cholesterol efflux function.

<p>(A) Cell toxicity of GW3965. Huh7.5 cells were cultured in the presence of indicated concentrations of the drug for 24 h. The luminescent signal is expressed in luminescence units (RLU). (B) Up-regulation of ABCA1 mRNA expression by GW3695 treatment. Huh7.5 cells were treated for 24 h with 1 µM GW3695 or drug solvent (DMSO). Then ABCA1 mRNA was determined by qRT-PCR. (C) ABCA1 protein production in drug-stimulated Huh7.5 cells. Cells were treated for 24 h with 1 µM GW3965 and analysed by Western blot (shown in the insert). Protein content in the ABCA1 band (220 kDA) in GW3965-(GW), and DMSO-(solv) treated cells was quantified relative to the calnexin band using the Odyssey Infrared Imaging System. (D) GW3965 stimulation promotes ABCA1-mediated cholesterol efflux to ApoA1. Huh7.5 cells were labelled with [³H] cholesterol then incubated with GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed by comparing cell-associated and free radioactivity. (E) Kinetics of ABCA1 gene expression following stimulation of cells with GW3965. Huh7.5 cells were treated with 1 µM GW3965 for the indicated time and ABCA1 mRNA was determined by qRT-PCR. Results were expressed as relative values compared to ABCA1 expression in cells treated with drug solvent. (F) Kinetics of cholesterol efflux in cells stimulated with GW3965. Huh7.5 cells were labelled with [³H] cholesterol for 24 h, and incubated for an additional 16 h with 1 μM GW3965 or drug solvent. ABCA1-dependent [³H] cholesterol efflux was assayed in the presence of ApoA1 and either GW3965 or solvent for the indicated period of time.</p

Up-regulation of ABCA1 inhibits HCV cell entry.

<p>The effect of GW3965 on the HCV cell cycle was analysed by adding the drug at different time points. A flow-chart is depicted in the upper panel of the graph. RNA in Huh7.5 cells infected in the presence of DMSO is shown in (a); that in cells pre-treated for 24 h with 1 µM GW3965 and infected in the presence of the drug are shown in (b), (c) and (d); results for cells treated with GW3965 during virus inoculation without pre-treatment are shown in (e); those of assays where the drug was added at 2 h, 4 h, or 6 h post-infection are presented in (f), (g) and (h) respectively. For each experiment cells were incubated for the indicated time period after infection (IV). The efficiency of infection was expressed as intracellular HCV RNA measured by qRT-PCR as a per cent of the control (a).</p

Analysis of HCV particles secreted from cells that over-express ABCA1.

<p>Physical properties of the nascent virus particles produced in cells stimulated or not with GW3965 were analysed by centrifugation in iodixanol gradient. Huh7.5 cells were pre-incubated with solvent (panel A) or 1 µM GW3965 (panel B) and the drug was maintained until 72 h post-infection when cell supernatants were collected, concentrated and subjected to gradient centrifugation. HCV RNA in gradient fractions was quantified by qRT-PCR and core antigen, ApoB and ApoE by ELISA assays.</p

Over-expression of ABCA1 inhibits HCV infection of primary human hepatocytes and human liver slices.

<p>(A–B) Inhibition of HCV infection of primary human hepatocytes. (A) Primary human hepatocytes were treated with 2–10 μM GW3965 (non-toxic concentrations for cells) or with drug solvent, prior to HCV infection. Twenty-four hours post-infection, ABCA1 mRNA was determined by qRT-PCR and expressed in arbitrary units, taking into account ABCA1 levels in liver cells pre-treated with the drug. (B) GW3965-treated and solvent-treated primary human hepatocytes were inoculated with HCV. After 24 h, intracellular HCV RNA was quantified by qRT-qPCR. The efficiency of infection in drug pre-treated cells was expressed as the percentage of infection compared to solvent-treated cells. (C–D) ABCA1 over-expression inhibits HCV infection of human liver slices. Human liver slices were cultured for 24 h, treated with 5 or 10 μM GW3965 or with DMSO before infection with HCVcc. At 24 h post-infection, total RNA was extracted and ABCA1 mRNA (C) and HCV RNA (D) were quantified by corresponding qRT-PCR assays and expressed as the percentage of RNA compared to the values obtained for solvent-treated cells.</p