Most strongly up- and down-regulated genes in SDHD-ESR tissues.

a<p>; Values >0 indicate up-regulated genes. Values <0 indicate down-regulated genes. FDR: False discovery rate.</p

Biological functions predicted to be affected in the SDHD-ESR kidney.

<p>Biological functions predicted to be affected in the SDHD-ESR kidney.</p

Validation and tamoxifen dose-response characterization of the inducible SDHD-ESR mouse.

<p>A. Relative amount of <i>SdhD</i> functional alleles (<i>SdhD</i>⁺ or <i>SdhD^flox</i>) in kidney and liver as determined by quantitative PCR of genomic DNA prepared 3 weeks after the start of injections of 100 µg/g, 4 times (high tx); or 50 µg/g, 2 times (low tx) tamoxifen. B. Succinate-ubiquinone oxidoreductase activity (SQR) of mitochondrial complex II (MCII) and NADH-dehydrogenase (NDH) activity of mitochondrial complex I (MCI) in kidney and liver 3 weeks after the first tamoxifen injection. C. Growth curves of 7-week-old mice after treatment with the same doses of tamoxifen. D. Survival curves of animals treated with the low dose of tamoxifen. Control group includes homozygous (+/+) and heterozygous (+/−) individuals without CRE recombinase, as no differences in the tested phenotypes were found between the two genotypes. Between 3 and 8 individuals per group were analyzed in each experiment. *, <i>P</i>≤0.05; **, <i>P</i>≤0.01; ***, <i>P</i>≤0.001.</p

“Pseudo-hypoxic” response in SDHD-ESR mouse tissues.

<p><b>A.</b> Succinate-ubiquinone oxidoreductase activity (SQR) in kidney 7 days after the start of the tamoxifen treatment. <b>B.</b> Relative mRNA level of <i>Vegf</i>, <i>Glut1</i>, and <i>Phd3</i> genes in kidney at 7 and 21 days after the start of the tamoxifen treatment. n.d.: non-determined. <b>C.</b> Western blot of Hif1α with protein extracts from kidney after tamoxifen treatment. Protein extracts from the pancreas of a β-cell-specific von Hippel-Lindau gene knock-out (VHL-KO) mouse and a wild-type littermate (VHL-wt) are loaded as controls. n.a.: non-applicable. Between 3 and 8 individuals per group were analyzed in each experiment. *, <i>P</i>≤0.05; ***, <i>P</i>≤0.001.</p

Functional analysis and validation of the microarray.

<p><b>A.</b> Comparative functional analysis of datasets generated from adrenal medulla (AM) and kidney samples from SDHD-ESR mice. The significance of each molecular and cellular function is indicated by –log of the p-value. <b>B.</b> Quantitative RT-PCR of RNA samples used in the microarray study based on specific primers for amplification of the <i>Cdkn1a</i> mRNA.</p

Microarray analysis.

<p><b>A. </b><i>SdhD</i> mRNA levels in heterozygous (+/−) and SDHD-ESR adrenal medulla and kidney relative to wild-type (+/+) tissues 7 days after the start of the tamoxifen treatment, as obtained from the corresponding microarray feature (Gene Bank accession n° NM_025848). *, <i>P</i>≤0.05; ***, <i>P</i>≤0.001. The number of samples is 8 per group. <b>B.</b> Supervised hierarchical clustering of adrenal medulla (AM) and kidney samples based on genes that showed significant differences in their expression level. The heatmap and the hierarchical tree are shown for 8 samples, grouped in pairs, per genotype.</p

Expression levels of HIF1α-mediated hypoxia responsive genes.

<p>Date are expressed as the log ratio ± SEM between either the homozygous (+/−) or the inducible SDHD-ESR mutant and the wild type (+/+) expression levels for each gene in each tissue as obtained from the microarray analysis. <i>Glut1</i>: glucosyltransferase 1 (NM_172380), <i>HK2</i>: Hexokinase 2 (NM_013820), <i>LDHA</i>: Lactate dehydrogenase A (NM_001136069), <i>PDK1</i>: Pyruvate dehydrogenase kinase 1 (NM_172665), <i>Vegf</i>: Vascular endothelial growth factor (NM_001025257).</p

Analysis of p21^WAF/Cip in SDHD-ESR-derived cell lines.

<p><b>A, B.</b> Western blot of p21^WAF1/Cip1 in total protein extracts of (<b>A</b>) MEFs and (<b>B</b>) BMK cells obtained from SDHD-ESR mice and their homozygous <i>SdhD^flox</i>^/+ (+/+) and heterozygous <i>SdhD^flox/−</i> (+/−) littermates and cultured in medium supplemented with 4-hydroxy-tamoxifen for 4 or 24 hours. Quantification of relative p21^WAF1/Cip1 band intensities in (<b>C</b>) MEFs and (<b>D</b>) BMK cells normalized to β-actin signal. Results are the average ± SEM of three independent experiments. Two different immortalized clones were generated for each genotype and cell type giving the same results. *, <i>P</i>≤0.05; ***, <i>P</i>≤0.001; for 0 hours, i.e. in the absence of 4-hydroxy tamoxifen, versus 4 or 24 hours of incubation in 4-hydroxytamoxifen.</p

Overlay of the X-ray diffraction models for the WT and mutant TAX-HLA-TCR complexes.

<p>All the panels show an overlay of the Cα atoms of the TAX-HLA-TCR complex (pdb: 1ao7) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154219#pone.0154219.ref015" target="_blank">15</a>] with those corresponding to V7R (pdb: 1qse) P6A (1qrn) and Y8A (pdb: 1qsf) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154219#pone.0154219.ref022" target="_blank">22</a>]. A) Overlay of the full complex. TCR molecules are in black lines. Black ball-and-sticks represent the antigenic peptide. The HLA domain containing the peptide is in dark grey lines and the rest of the structure in light grey. B) detail of the ternary complex interface for the structure overlay in (A). C) Apical view of TCR and the antigenic peptide in the above overlay of the four structures. As in (A), TCR atoms are represented by lines, but those from residues closer than 6 Å from HLA are highlighted by stick representation. D) Apical view of HLA and the antigenic peptide for the four structures in the overlay. HLA residues closer than 6 Å from TCR are highlighted in a ball-and-stick representation.</p

Vibrational entropy contributions (-TΔS^conf) to binding-free energy.

<p>Vibrational entropy contributions (-TΔS^conf) to binding-free energy.</p