Antiviral activity of Bay 41-4109 on HBV replication in HepG2.2.15 cells.

<p>Cells were treated with Bay 41-4109 (25, 50, 100, 200 and 400 nM) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025096#s2" target="_blank">Methods</a> section. HBV DNA in HepG2.2.15 supernatant was quantified by real-time PCR. The data represent the results of three independent experiments, performed in duplicate.</p

Generation of MITF-A transgenic mice.

<p><b>A)</b> Schematic representation of the Ksp-cadherin-FLAG-MITF-A transgene. <b>B)</b> <i>Mitf-A</i> mRNA expression evaluated by quantitative RT-PCR in kidneys from wild-type (WT), heterozygous (HE) and homozygous (HO) MITF-A transgenic mice (line 42) 2 months after birth. Data are means ± SEM; n = 4–6 per each genotype. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: ** P < 0.01, *** P < 0.001. <b>C)</b> MITF-A protein expression evaluated by western blot on kidney nuclear protein extracts from WT, HE and HO MITF-A transgenic mice 2 months after birth. This is a representative image of three experiments. Nuclear protein extracts from <i>Mitfa</i>^-/- kidneys were used as a negative control; crude extracts from renal cells transfected with either FLAG-MITF-A plasmid (lane 1) or MITF-A plasmid (lane 2) were used as a positive control. Lamin A/C was used as control of nuclear protein amount. IB = immunoblot.</p

HBV viral load in control and treated infected uPA chemeric mice.

1<p>an arbitrary threshold of detection of 100 copies/ml was used for calculation.</p>2<p>nonparamertic Mann-Whitney test.</p

Immunohistological analysis of human hepatocytes in mouse livers.

<p>Serial sections of chimeric mouse livers from treated (T529 and T490) and untreated (C371) mice were analyzed by labeling with anti-α1 anti-trypsin (αAT) or with anti-HBc (HBc) antibodies.</p

Antiviral activity of Bay 41-4109 on HBV replication in humanized Alb-uPA/SCID mice.

<p>(A) Human albumin concentrations in sera from treated (full lines) and untreated (dotted lines) animals. (B) The HBV viral load during the course of the experiment was quantified by real-time PCR in sera from treated (full lines) and untreated (dotted lines) animals. (C) Histograms represent the mean HBV load ratio at specific time points in each treated (white) and untreated (black) animal. Data are represented on semilogarithmic graphs.</p

Characterization of MITF-A transgenic mice.

<p>Characterization of MITF-A transgenic mice.</p

Expression pattern of MITF-A during kidney development.

<p><b>A-B)</b><i>In situ</i> hybridization of <i>Mitf-A</i> of E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos using an antisense RNA probe directed against a sequence encompassing exon 1A, specific for <i>Mitf-A</i>, and exon 1B common to <i>Mitf-A</i>, <i>Mitf-H</i>, <i>Mitf-C</i>, <i>Mitf-J</i> and <i>Mitf-Mc</i> isoforms. The inset shows the staining of E13.5 kidneys using the sense RNA probe. Magnifications are X100 (left panels), X200 (middle panels) and X400 (right panels). In WT kidneys <b>(A)</b> a weak staining is observed in branches of UB (black arrow), in S-shaped body (blue arrow) and in metanephric mesenchyme (asterisk). Consistent with the use of the Ksp-cadherin promoter, the signal in MITF transgenic kidneys <b>(B)</b> was strongly increased in UB and tips (black arrow), in ureteric tip (black arrow) and to a lesser extent in S-shaped body (blue arrow). <b>C)</b> <i>In situ</i> hybridization of <i>Mitf-A</i> in transgenic HO kidneys after laminin immunohistochemistry (red). Note <i>Mitf</i> expression in ureteric bud and tip (black arrow), in and S-shaped body (blue arrow). Magnification X400. Sections are representative images of 4 kidneys per genotype. <b>D</b>) Immunostaining of MITF-A in WT and HO MITF-A transgenic metanephroi at E13.5. Note the increase of MITF-A expression in UB stalks, tips and S-bodies. Magnification X400.</p

Impact of MITF-A overexpression on cell survival.

<p><b>A-B)</b> Cell proliferation in E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos. Proliferating cells were identified using an anti-phospho-histone H3 (pH3) <b>(A)</b> and an anti-PCNA antibody <b>(B)</b>. Magnifications are X400 and X600, respectively. Left panels: representative images of 5 kidneys; right panels: quantification of the number of pH3-positive and PCNA-positive cells per UB structure. <b>C)</b> Apoptosis was evaluated by TUNEL assay in E13.5 kidneys from WT and HO MITF-A transgenic embryos. Left panels: representative images of 5 kidneys (magnification X400); right panels: quantification of the number of TUNEL-positive cells per microscopic field. Data are means ± SEM. Quantifications were performed on three sections for each kidney (n = 5 mice per genotype). Mann-Whitney test; transgenic <i>versus</i> wild-type mice: *** <i>P</i> < 0.001.</p

<i>Mitfa</i> inactivation results in reduced glomeruli number.

<p><b>A)</b> Schematic representation of the targeting strategy used to inactivate <i>Mitfa</i>. <b>B-C)</b> <i>Mitf-A</i> <b>(B)</b> and total <i>Mitf</i> mRNA <b>(C)</b> expression evaluated by quantitative RT-PCR in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. <b>D)</b> Glomerular number in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. Data are means ± SEM, n = 8–10 per each genotype. Mann-Whitney test; <i>Mitfa</i>^<i>-/-</i> <i>versus Mitfa</i>^<i>+/+</i>: *** P < 0.001.</p

Expression of candidate MITF-A targets in E13.5 kidneys.

<p><b>A)</b><i>In situ</i> hybridization of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> in wild-type (WT) and homozygous (HO) MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). <b>B)</b> Quantitative RT-PCR analysis of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> mRNA expression in E13.5 kidneys of WT, heterozygous (HE) and HO MITF-A transgenic embryos (n = 6–9 per genotype). <b>C)</b> <i>In situ</i> hybridization of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> in WT and HO MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). Note the increased staining of <i>Re</i>t mRNA in transgenic kidneys at E13.5. <b>D)</b> Quantitative RT-PCR analysis of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> mRNA expression in E13.5 kidneys of WT, HE and HO MITF-A transgenic embryos (n = 6–9 per genotype). Data are means ± SEM. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: * P < 0.05, ** P < 0.01.</p