Additional file 4: of Identification of new loci involved in the host susceptibility to Salmonella Typhimurium in collaborative cross mice

Figure S3. Founder contributions and haplotype around Stsl4 QTL on Chr 6. (A) Genome scan magnification for Stsl4 QTL region (60–100 Mb on Chr 6). The mouse genome location is on the X-axis and significance (−log10(P)) values on the Y-axis, with genome-wide thresholds of association at E < 0.5, E < 0.1 and E < 0.05 levels indicated respectively by the gray, orange and red lines. Peak location (maximum value of –log10(P)) is marked by a star. (B) Founder contributions in the same magnified region. The peak location is marked by a star. Each of the 8 founders is in a different color. The mouse genome location is on the X-axis and Y-axis shows the founder estimated effect on splenic bacterial load after S. Typhimurium infection. (C) Founder contributions at Stsl4 QTL peak (81.2 Mb). X-axis shows the different founder strains. Y-axis shows the estimated founder effect. No obvious contributions explain Stsl4 QTL, but B6 has the lowest estimated impact while NZO/HILtJ and PWK/PhJ have the highest estimates. (PDF 160 kb

Additional file 6: of Identification of new loci involved in the host susceptibility to Salmonella Typhimurium in collaborative cross mice

Table S2. Genes remaining in Stls1 interval post merge analysis. Gene symbol, start and end positions, name, high merged SNPs, expression in immune cell, cell-type major expression and Gene Ontology (GO) terms are given. Gene positions (build mm9), names as well as GO terms were collected from UCSC, MGI and ENSEMBL, while expression data were collected from Male/Female RNAseq of ImmGen. (XLSX 496 kb

Additional file 7: of Identification of new loci involved in the host susceptibility to Salmonella Typhimurium in collaborative cross mice

Table S3. Genes remaining in Stls2 interval post merge analysis. Gene symbol, start and end positions, name, high merged SNPs, expression in immune cell, cell-type major expression and Gene Ontology (GO) terms are given. Gene positions (build mm9), names as well as GO terms were collected from UCSC, MGI and ENSEMBL, while expression data were collected from Male/Female RNAseq of ImmGen. (XLSX 492 kb

Additional file 3: of Identification of new loci involved in the host susceptibility to Salmonella Typhimurium in collaborative cross mice

Figure S2. Founder contributions and haplotype around Stsl3 QTL on Chr 1. (A) Genome scan magnification for Stsl3 QTL region (70–100 Mb on Chr 1). The mouse genome location is on the X-axis and significance (−log10(P)) values on the Y-axis, with genome-wide thresholds of association at E < 0.5, E < 0.1 and E < 0.05 levels indicated respectively by the gray, orange and red lines. Peak locations Stsl3a and Stsl3b (maximum value of –log10(P)) are marked by stars. (B) Founder contributions in the same magnified region. The peak location of Stsl3a is marked by a star. Each of the 8 founders is in a different color. The mouse genome location is on the X-axis and Y-axis shows the founder estimated effect on splenic bacterial load after S. Typhimurium infection. (C) Founder contributions at Stsl3a QTL peak (83.9 Mb). X-axis shows the different founder strains. Y-axis shows the estimated founder effect. No obvious contributions explain Stsl3a QTL, but B6 (grey) has the highest estimated impact of the 8 founders. (D) Founder contributions at Stsl3b QTL peak (79.2 Mb). There is no obvious founder contribution for Stsl3b QTL peak region. 129 (pink) has the highest estimated impact of the 8 founders while PWK (red) has the lowest estimate. (PDF 215 kb

Survival study and kidney injury in 129S2/SvPasCrl and C57BL/6J mice.

<p><b>A</b>, Survival proportions of mice from both strains before sacrifice at 16 weeks. 129S2/SvPasCrl mortality was significantly increased (p = 0.045) and necropsy revealed kidney dilation due to urolithiasis. <b>B</b>, Infrared cartography of 129S2/SvPasCrl kidney slices revealed the presence of focal cystine aggregates in renal tissues (cystine tubular casts). <b>C</b>, Renal function as assessed by enzymatic serum creatinine dosage was not significantly impaired in 129S1/SvPasCrl mice (n = 6) compared to C57BL/6J mice (n = 9) at 16 weeks (p = 0.11). <b>D–F</b>, Macrophage infiltrate in kidney tissues was assessed by morphometric analyses of F4-80 immunostaining and was increased in 129S2/SvPasCrl mice in comparison with C57BL/6J (n = 5/group, p = 0.046). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">Figures 3E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g003" target="_blank">3F</a> are representative of macrophage infiltrates in C57BL/6 and 129S1/SvPasCrl mice respectively (magnification x200+ zoom). <b>G</b>, Fibrosis assessed by sirius red morphometric analysis did not evidence significant fibrosis amount in both strains (percentage of fibrotic area, p = NS).</p

Identification of a functional missense mutation in a highly conserved sequence of the <i>Slc3a1</i> gene in 129S2/SvPasCrl mouse.

<p><b>A</b>, Mutation of G by A nucleotide at position 1232 in 129S2/SvPasCrl mouse (c.1232G>A). <b>B</b>, In position 383, the glutamine is substituted by a lysine in the extracellular part of rBAT protein in 129S2/SvPasCrl mouse. The sequence is highly conserved among mammal species. <b>C</b>, 129S1/Sv mice from the Jackson laboratory express rBAT at the brush border of proximal tubular cells. <b>D</b>, Eighty percent of 129S2/SvPasCrl mice sacrificed at 10 weeks were affected by urolithiasis whereas no 129S2/SvPas (G/G genotype) mouse was affected (n = 5/group, p = 0.047). <b>E</b>, One hundred percent of 129S2/SvPasCrl mice presented cystine crystals in urine whereas no 129S2/SvPas (G/G genotype) mouse was affected (n = 5/group, p = 0.008). <b>F</b>, Urinary aminoacid chromatography has been performed in 129S2/SvPasCrl mice and 129S2/SvPas mice (n = 5/group). Cysteine (reduced form of cystine) was significantly higher in 129S2/SvPasCrl mice urine (p = 0.008). Results are expressed as aminoacid concentration (µMol)/creatinine concentration (mMol) in urines.</p

mRNA and protein expression of cystine transporters <i>Slc3a1</i>/rBAT and <i>Slc7a9</i>/b^0,+AT in kidney cortex from 129S2/SvPasCrl and C57bBL/6J mice.

<p><b>A–B</b>, Quantitative PCR: <i>Slc3a1</i> and <i>Slc7a9</i> mRNA expression was similar in both strains. <b>C</b>, Western Blot: b^0,+AT was expressed at a similar level in kidney cortex from both strains. <b>D</b>, Western Blot: rBAT was expressed in C57BL/6 mice but not in 129S2/SvPasCrl mice. <b>E–H</b>, Antibodies directed against the extracellular part of rBAT (Figures 4E and 4G) or against its intracellular part (Figures 4F and 4H) revealed the presence of rBAT at the brush border of proximal tubular cells in C57BL/6J mice (4E and 4F) but not in 129S2/SvPasCrl mice (4G and 4H).</p

129S2/SvPasCrl crystalluria and aminoaciduria are similar to those of human patients with cystinuria.

<p><b>A–B</b>, Typical flat hexagonal cystine crystals were present in 129S2/SvPasCrl fresh urine. <b>C</b>, Ninety percent of 129S2/SvPasCrl mice presented crystalluria (p = 0.0001 vs C57BL/6J mice). <b>D</b>, Urinary aminoacid chromatography has been performed in 129S2/SvPasCrl mice (n = 5) and C57BL/6J mice (n = 3). Dibasic aminoacids including cysteine (reduced form of cystine) were significantly higher in 129S2/SvPasCrl mice urine (p = 0.036 for each dibasic aminoacid) whereas other aminoacid renal excretion was similar in both strains. Some representative aminoacids are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102700#pone-0102700-g001" target="_blank">Figure 1D</a>. Results are expressed as aminoacid concentration (µMol)/creatinine concentration (mMol) in urines.</p

129S2/SvPasCrl mouse is affected by cystine urolithiasis.

<p><b>A</b>, Eighty percent of 129S2/SvPasCrl mice sacrificed at 16 weeks or dead before 16 weeks have been affected by urolithiasis whereas no C57BL/6J mice has been affected, p = 0.0007, n = 10/group. <b>B</b>, Bladder stones in a 129S2/SvPasCrl mouse. <b>C</b>, Spectrum of cystine was obtained by FTIR analysis of mouse stones. <b>D–F</b>, Scanning electron microscopy of a stone, at x58, x506, and x1070 magnification respectively, revealed the typical flat hexagonal structure of cystine crystals.</p