Table of primers.

<p>Table of primers.</p

Hepatic stellate cells are responsive to stimulation by TLR ligands.

<p>(A) mRNA level of IL-6 was quantified by qRT-PCR in four separate preparations of activated rat HSCs (culture day 10) treated with TLR2 ligand (LTA, 100 ng/ml), TLR3 ligand (Poly(I∶C), 1 µg/ml), TLR4 ligand (LPS, 100 ng/ml) or TLR5 ligand (Flagellin, 1 µg/ml) for up to 24 hours (n = 4). Expression level was normalised to β actin. (B) Secreted IL-6 protein was measured by ELISA in conditioned media collected from activated HSCs treated with TLR ligands as in (A) following 2 h, 8 h or 24 h of stimulation (n = 4). (C) mRNA levels of IL-6, TIMP1, αSMA and collagen I were quantified by qRT-PCR in four separate preparations of activated rat HSCs (culture day 10) treated with TLR3 ligand (Poly(I∶C), 1 µg/ml) for up to 24 hours. Expression level was normalised to β actin. (#p<0.1, *p<0.05, **p<0.01***p<0.001).</p

Interferon gamma and IL-6 expression are TLR3 mediated and loss of ability to induce IFNγ in aHSCs is associated with transcriptional repression by Polycomb complex.

<p>(A) WT and <i>tlr3</i>^−/− activated mHSCs were treated with Poly(I∶C) (1 µg/ml) for 8 and 24 hours; subsequently IFNγ mRNA expression was measured by qPCR. Data are normalised to GAPDH and expressed as fold induction relative to control. (B) IL-6 mRNA expression was measured in WT and <i>tlr3</i>^−/− activated mHSCs in response to increasing concentrations of Poly(I∶C). (C) Wild type qHSCs were treated with transcriptional inhibitors Act D (1 µM) and DRB (80 µM) for 24 hours and IL-6 mRNA expression analysed by qRT-PCR. Data are normalised to GAPDH and expressed as fold induction relative to untreated control. (D and E) One hundred micrograms of crosslinked chromatin from quiescent and activated rat HSC was incubated with 10 µg of anti-trimethyl and anti-dimethyl H3K27 antibody and ChIP assay performed. Data are expressed as fold enrichment relative to IgG control. (*p<0.05, **p<0.01, ***p<0.001).</p

Activation of HSC decreases TLR3 mediated interferon and cytokine response.

<p>(A,B,C) HSCs were isolated from control, acute CCl₄ treated rats (single injection), or chronic CCl₄ treated rats (4 weeks twice weekly injections); cells were seeded onto plates and treated with Poly(I∶C) (1 µg/ml) for up to 24 hours. Interferon α, β and γ were measured and normalised to β actin. Data are expressed as RLTD. (D) Interferon-inducible cytokines CXCL10, TNF-α, MxA, CXCL1/KC, IL-1β and indoleamine 2,3-dioxygenase were measured and normalised to β actin (n = 4). Data are presented as fold change, Poly(I∶C) stimulated to unstimulated. Secreted IFNγ was measured by ELISA in the media of cultured HSCs isolated from rats treated with either chlodronate-liposomes (E) or empty liposome control (F). (G) Expression of MHC class II on macrophages cultured in qHSC conditioned media. Flow cytometric analysis of control macrophages (untreated), or macrophages incubated with control media, untreated qHSC conditioned media, Poly(I∶C) treated qHSC conditioned media or 100 ng/ml recombinant IFNγ as positive control. (*p<0.05, **p<0.01, ***p<0.001).</p

Quiescent and activated HSCs express Toll like receptors.

<p>(A) mRNA levels of TLR1-13 were quantified by qRT-PCR in three separate preparations of primary rat qHSCs (day 0) and day 10 transdifferentiated myofibroblasts. Data are expressed as relative level of transcriptional difference (RLTD) to TLR1 mRNA expression (n = 3). (B) Thirty micrograms of whole cell protein extract from three separate preparations of quiescent rat HSCs (culture day 1) or activated myofibroblasts (culture day 10) were separated by SDS-PAGE and immunoblotted for TLR3 and GAPDH. (C) Toll-like receptor 3 was visualised in the cytoplasm of rat qHSCs (<i>ex vivo</i>) culture day 1 (bar represents 75 µm). (D) Quiescent rat HSCs were treated with Poly(I∶C) (1 µg/ml) or IL-1α (2 ng/ml) for up to 24 hours; IL-6 mRNA was measured and normalised to β actin (n = 3). (E) Thirty micrograms of whole cell protein extract from two separate preparations of quiescent HSCs or activated myofibroblasts (culture day 10) were separated by SDS-PAGE and immunoblotted for IRAK1, TRAF6 and GAPDH. (F) Activated rat myofibroblasts were treated with IL-1α (2 ng/ml) for up to 24 hours; IL-6 and TIMP1 mRNA were measured and normalised to β actin (n = 3). (*p<0.05, ***p<0.001).</p

Gene expression analysis in NB and HeLa cells and correlation analysis.

<p>(A) Relative gene expression analysis of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes, carried out in a panel of NB and in HeLa cell lines by real-time RT-qPCR using a pool of normal tissue RNAs as reference sample (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#s4" target="_blank">Methods</a>), shows over-expression of the three genes in all but two NB cell lines tested (GI-ME-N and ACN). (B) X-Y Plots showing a significant correlation between the expression level of <i>PHOX2B</i> and <i>PHOX2A vs.ALK</i> (left) and <i>PHOX2Avs. PHOX2B</i> (right) genes in the analyzed cell lines. Pearson's correlation coefficient indicates a very significant correlation of the three transcription levels <i>vs.</i> each other. Values are the mean ± s.d. of N = 3 independent RT-qPCR analyses performed in triplicate.</p

Sequences of primers employed for real-time RT-qPCR.

<p>Sequences of primers employed for real-time RT-qPCR.</p

PHOX2B effect on the <i>ALK</i> promoter sequentially deleted plasmids.

<p>A) Schematic representation of deleted plasmid inserts, progressively shorter from the entire wt <i>ALK</i> promoter region considered (−671 bp), down to the so called deletion 2 (del2; −351 bp), and to the so called deletion 3 (del3; −31 bp) (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#pone-0013108-g004" target="_blank">figure 4A</a>). The promoter (grey bar), the 5′UTR (black bar) and the ATTA boxes (black circles) are shown. B) Activity of the <i>ALK</i> promoter fragments, expressed as percentage of the activity of the wt construct. Values are the mean ± s.d. of N = 3 independent experiments performed in HeLa cells. C) PHOX2B-mediated induction of the <i>ALK</i> promoter deleted plasmids, expressed as fold increase of the Luciferase activity obtained with respect to the use of the empty vector (pcDNA3.1) on the wt promoter. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate in HeLa cells.</p

Forced over-expression of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in HeLa and NB cell lines.

<p>A) Transcription levels of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes were evaluated in HeLa cells 48 hours post-transfection with the corresponding gene-specific cDNA expressing vectors by real-time RT-qPCR. Besides the dramatic increase of gene transcripts by each respective transfectants, <i>ALK</i> expression results enhanced by the <i>PHOX2</i> genes over-expression. Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. B) Upper (I) and lower (II) lanes from immunofluorescence analysis report examples of HeLa cells transfected with the PHOX2B-Myc expression construct. From left to right images show DAPI stained cell nuclei (blue), staining for the PHOX2B-Myc protein (green), and staining for the ALK protein (red). The most distal image is the merge of the three nearby figures. C) Western blot evaluating protein amounts of ALK and PHOX2B in HeLa cells at 72 hours post-transfection with gene-specific cDNA constructs. A consistent transcript increment of each gene is observed but ALK starts to be expressed also following the forced expression of PHOX2B-Myc protein. Gel was loaded with 100 µg of total protein extracts except for evaluation of ALK in <i>ALK</i>-transfected cells and PHOX2B in <i>PHOX2B</i>-transfected cells, for which 1∶10 (10 µg) of protein extracts was loaded to avoid over-saturation of autoradiograph films. D) Western blot evaluating protein amounts of ALK in ACN cells (left) at 72 hours post-transfection with the PHOX2B-Myc construct, in a clone of IMR-32 cells stably expressing the same PHOX2B-Myc fusion protein (right). A marked increment in ALK expression is detected following PHOX2B-Myc expression in transient –transfected ACN cells compared to both native and mock-transfected cells and in a clone of IMR-32 cells, stably expressing PHOX2B-Myc, compared to native cells. An anti-cMyc antibody was specifically used to distinguish PHOX2B-Myc fusion protein from endogenous PHOX2B of NB cells (lower blots). E) Two stable IMR-32 clones, one negative (104) and one positive (49) for PHOX2B-Myc expression were analyzed for ALK expression (upper panels); expression of the fusion PHOX2B-Myc protein was assessed by Western Blot using anti –cMyc (middle panels) and the anti-actin antibodies (bottom panels).</p

Effect of ATTA 4/5 disruption in competition of PHOX2B binding.

<p>IMR32 nuclear extracts were incubated with the ATTA4/5 probe (lane 2) and competition obtained by adding an excess of: a wild type (wt) probe (lane 3), a probe mutated in both ATTA 4 and ATTA 5 sites (lane 4) or in each of them (lanes 5–6). Incubation without nuclear extracts was regarded as negative control (lane 1).</p