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

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

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

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

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

“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

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