Additional file 1: of Characterization of interactions between hepatitis C virus NS5B polymerase, annexin A2 and RNA – effects on NS5B catalysis and allosteric inhibition

Figure S1. No Interactions between AnxA2 and NS3 or NS3/4A were observed by SPR. Figure S2. No Influence of AnxA2 upon enzyme activity of NS3. Table S1. Michaelis constant (KM) and maximal velocity (Vmax) for NS3 enzyme activity with and without the presence of AnxA2, derived from a FRET enzyme activity assay (Figure S2). (DOC 237 kb

AnxA2-D_I and AnxA2-D_IV do not bind to α-actin.

<p>∼10 µg of AnxA2-D_I (∼55 µM) (lane 1), AnxA2-D_IV (∼55 µM) (lane 2) and 5 µg of AnxA2 (∼6 µM) (lane 3) were separated by 15% SDS-PAGE (<b>A</b> and <b>B</b>) and transferred to a nitrocellulose membrane (<b>B</b>). Proteins were visualised by Coomassie Brilliant Blue staining (<b>A</b>). Far-Western (<b>B</b>); after denaturation and renaturation as described in Methods, the proteins were subjected to an actin overlay assay by incubation ON with 10 µg/ml α-actin and subsequent detection of bound actin by monoclonal actin antibodies. The positions of full-length (FL) AnxA2, and the domains I and IV of AnxA2 are indicated by arrowheads to the right. Selected standards are indicated by arrowheads to the left.</p

The effects of soluble AnxA2-D_I, AnxA2-D_IV and lysozyme on the formation of an <i>in vitro</i> capillary-like network.

<p>Co-cultures of SMCs and GFP-expressing HUVECs were treated with 5–15 µM AnxA2-D_I (<b>B</b>–<b>D</b>), AnxA2-D_IV (<b>G</b>–<b>I</b>), lysozyme (<b>L</b>–<b>N</b>) at 2 h after seeding. Panels <b>A</b>, <b>F</b> and <b>K</b> show the corresponding controls with untreated cells. After 72 h incubation, images were taken at 10× magnification. The tube total length was analysed (<b>E</b>, <b>J</b> and <b>O</b>) and expressed as percentage relative to the corresponding untreated EC controls (100%) (<b>A</b>, <b>F</b> and <b>K</b>, respectively), as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060281#pone-0060281-g001" target="_blank">Figure 1</a>. Results (<b>E</b>, <b>J</b> and <b>O</b>) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).</p

Domains I and IV of Annexin A2 Affect the Formation and Integrity of <i>In Vitro</i> Capillary-Like Networks

<div><p>Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an <i>in vitro</i> co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.</p> </div

The effect of monoclonal AnxA2 antibodies on the formation of an <i>in vitro</i> capillary-like network.

<p>Co-cultures of SMCs and GFP-expressing HUVECs were untreated (<b>A</b>), or treated with 4 µg/200 µl monoclonal AnxA2 antibodies (BD Biosciences) (<b>B</b>), (C-10; Santa Cruz), (<b>D</b>), Tyr23 AnxA2 specific antibodies (Santa Cruz) (<b>F</b>) or normal mouse IgG (<b>H</b>) at 2 h after seeding. After 72 h incubation, images were taken at 10× magnification. The tube total length was analysed (<b>C</b>, <b>E</b>, <b>G</b> and <b>I</b>) and expressed as percentage relative to the corresponding untreated EC control (100%) (<b>A</b>), as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060281#pone-0060281-g001" target="_blank">Figure 1</a>. Results (<b>C</b>, <b>E</b>, <b>G</b> and <b>I</b>) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).</p

The effects of AnxA2, S100A10 and the heterotetrameric AnxA2₂-S100A10₂ complex on the formation of an <i>in vitro</i> capillary-like network.

<p>Co-cultures of SMCs and GFP-expressing HUVECs were untreated (A), or treated with 15 µM AnxA2 (B), 15 µM S100A10 (C), 6 µM AnxA2₂-S100A10₂ complex (12 µM AnxA2) (D) or 100 nM PTK787 (E) at 2 h after seeding. After 72 h incubation, images were taken at 10× magnification. The tube total length was analysed (F) and expressed as percentage relative to the untreated EC control (100%) (A) using the Attovision and BD Image Data Explorer programmes. Results (F) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).</p

AnxA2-D_I and AnxA2-D_IV disrupt <i>in vitro</i> preformed capillary-like networks.

<p>Co-cultures with a preformed EC network were untreated (<b>A</b>, <b>D</b>, <b>G</b> and <b>J</b>), or treated for 3 h with 15 µM AnxA2-D_I (<b>B</b>, <b>E</b>, <b>H</b> and <b>K</b>) or AnxA2-D_IV (<b>C</b>, <b>F</b>, <b>I</b> and <b>L</b>). The cells were fixed in 3% paraformaldehyde and permeabilised with 0.05% Triton X-100 in PBS before processing for immunofluorescence using antibodies against endogenous AnxA2 (BD Biosciences). Note the filopodia-like structures (arrowheads) particularly in Panels <b>K</b> and <b>L</b>. Inserts in Panels <b>G</b>, <b>H</b> and <b>I</b> are magnified and shown in Panels <b>J</b>, <b>K</b> and <b>L</b>, respectively. Graphical representation (<b>D</b>–<b>F</b>) of the fluorescence intensity profiles determined for cross-sections of the cell as indicated in <b>A</b>–<b>C</b>. The orientation of the sections (from I to II) corresponds to intensity profiles from left to right in <b>D</b>, <b>E</b> and <b>F</b>. Bars, 100 µm (<b>A</b>–<b>C</b>) or 50 µm (<b>G</b>–<b>I</b>).</p

AnxA2 and VE-cadherin co-localise in endosome- and filopodia-like structures in sub-confluent HUVECs grown as a monolayer.

<p>The cells were fixed in 3% paraformaldehyde and permeabilised with 0.05% Triton X-100 in PBS before further processing for dual label immunofluorescence using antibodies directed against endogenous VE-cadherin (A) and AnxA2 (B). (C) shows the merged image. Several sites of VE-cadherin and AnxA2 co-localisation are indicated by arrowheads. Bar, 40 µm.</p

Tyr23 phosphorylated AnxA2 is present in the ECM of confluent HUVECs.

<p>HUVECs were incubated for 30 min in the absence (control) or presence of 50 µM PP2 before harvesting. The control fraction (ECM) was obtained in the presence of 200 µM ortho-vanadate to inhibit dephosphorylation. 100 µg of EGTA-released extracellular proteins were subjected to 10% SDS-PAGE and subsequently transferred to a nitrocellulose membrane for the detection of AnxA2 by Western blot analysis using monoclonal antibodies directed against pTyr23 AnxA2 (<b>A</b>) or AnxA2 (BD Biosciences) (<b>B</b>). Selected standards are indicated by arrowheads to the left. AnxA2 and actin (as a loading control) are indicated by an arrowhead and asterisk, respectively.</p

The effects of AnxA2, S100A10, AnxA2-D_I, or AnxA2-D_IV on <i>in vitro</i> preformed capillary-like networks.

<p>Co-cultures with a preformed EC network were treated with 15 µM AnxA2 (<b>B</b>), S100A10 (<b>C</b>), AnxA2-D_I (<b>D</b>), or AnxA2-D_IV (<b>E</b>). After 72 h incubation, images were taken at 10× magnification. The degree of disruption of the mature vascular network was quantified (<b>F</b>) as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060281#pone-0060281-g001" target="_blank">Figure 1</a>. The tube total length is expressed as percentage relative to the untreated EC control (100%) (<b>A</b>). Results (<b>F</b>) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).</p