these animals stopped gaining weight after administration of the drug, and with the higher dose, even lost weight after two weeks. Highdose tamoxifen-treated MedChemExpress LY-411575 mutants were sacrificed before showing evident signs of suffering. Low-dose tamoxifen-treated mutants, although surviving longer than the high-dose treated animals, still had a shorter average life-span than their wild-type littermates. Since the treatment with the low dose of tamoxifen casts doubt about the efficient deletion of SdhD in all the tissues, the rest of experiments shown here were performed with the high dose. Western Blot For protein preparation, tissues and cells were homogenized in ice-cold HEN buffer containing 1 mM Na3VO4, 0.2% IGEPAL CA-630 and 1% protease inhibitor cocktail. Homogenized samples were centrifuged for 30 min. at high speed in a microcentrifuge, after which protein containing supernatant was collected. Protein concentration was determined using a protein assay kit from Bio-Rad. From each sample, 50 mg of protein was separated by electrophoresis on SDSpolyacrylamide gels and electroblotted onto PVDF 6099352 membranes. Blots were incubated in blocking solution, followed by overnight incubation with the following antibodies: anti-HIF1a; anti-Glut1; antip21WAF1/Cip1; and anti-b-actin. The membranes were then washed with PBS-T and incubated with either a HRP conjugated goat anti-rabbit IgG antibody or HRP conjugated sheep anti-mouse IgG antibody. Antibody detection was performed with an enhanced chemiluminescence reaction. The “Pseudo-hypoxic Drive”is Partially Recapitulated in SDHD-ESR Tissues To address the possible activation of the “pseudo-hypoxic drive”mechanism, the expression of several HIF1a target genes was analyzed in wild-type, heterozygous and SDHD-ESR mutant tissues. We present here data obtained from kidney as this organ shows the most intense SdhD deletion of those analyzed. To prevent possible secondary effects due to the sustained lack of the gene in the animals three weeks after the start of the of treatment, we included in our analysis kidney samples obtained one week after the first tamoxifen injection. At this time point, SQR activity had already decreased considerably. The messenger RNA levels of vascular endothelial growth factor, glucose transporter 1, and prolylhydroxylase 3 genes were determined by RT-qPCR, Statistical Analysis Data are presented as mean 6 standard error. Statistical significance 6099352 was assessed by ANOVA with appropriate post-hoc analysis. For paired groups, either a Student’s t-test with a Levene test for homogeneity of variances in the case of normal distribution, or the nonparametric U-Mann Whitney test in the case of non-normal distributions, was applied. PASW18 software was used for statistical analysis. Statistical analysis of the microarray data was performed with the Multi-Experiment p21WAF1/Cip1 Overexpression in a SdhD Mouse Mutant 7 p21WAF1/Cip1 Overexpression in a SdhD Mouse Mutant down-regulated.Date are expressed as the log ratio 6 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. Glut1: glucosyltransferase 1, HK2: Hexokinase 2, LDHA: Lactate dehydrogenase A, PDK1: Pyruvate dehydrogenase kinase 1, Vegf: Vascular endothelial growth factor. doi:10.1371/journal.pone.0085528.t001 revealing a non-statistically significant trend towards an increase in heterozygous animals compared with wild-t these animals stopped gaining weight after administration of the drug, and with the higher dose, even lost weight after two weeks. Highdose tamoxifen-treated mutants were sacrificed before showing evident signs of suffering. Low-dose tamoxifen-treated mutants, although surviving longer than the high-dose treated animals, still had a shorter average life-span than their wild-type littermates. Since the treatment with the low dose of tamoxifen casts doubt about the efficient deletion of SdhD in all the tissues, the rest of experiments shown here were performed with the high dose. Western Blot For protein preparation, tissues and cells were homogenized in ice-cold HEN buffer containing 1 mM Na3VO4, 0.2% 18083779 IGEPAL CA-630 and 1% protease inhibitor cocktail. Homogenized samples were centrifuged for 30 min. at high speed in a microcentrifuge, after which protein containing supernatant was collected. 
Protein concentration was determined using a protein assay kit from Bio-Rad. From each sample, 50 mg of protein was separated by electrophoresis on SDSpolyacrylamide gels and electroblotted onto PVDF membranes. Blots were incubated in blocking solution, followed by overnight incubation with the following antibodies: anti-HIF1a; anti-Glut1; antip21WAF1/Cip1; and anti-b-actin. The membranes were then washed with PBS-T and incubated with either a HRP conjugated goat anti-rabbit IgG antibody or HRP conjugated sheep anti-mouse IgG antibody. Antibody detection was performed with an enhanced chemiluminescence reaction. The “Pseudo-hypoxic Drive”is Partially Recapitulated in SDHD-ESR Tissues To address the possible activation of the “pseudo-hypoxic drive”mechanism, the expression of several HIF1a target genes was analyzed in wild-type, heterozygous and SDHD-ESR mutant tissues. We present here data obtained from kidney as this organ shows the most intense SdhD deletion of those analyzed. To prevent possible secondary effects due to the sustained lack of the gene in the animals three weeks after the start of the of treatment, we included in our analysis kidney samples obtained one week after the first tamoxifen injection. At this time point, SQR activity had already decreased considerably. The messenger RNA levels of vascular endothelial growth factor, glucose transporter 1, and prolylhydroxylase 3 genes were determined by RT-qPCR, Statistical Analysis Data are presented as mean 6 standard error. Statistical significance was assessed by ANOVA with appropriate post-hoc analysis. For paired groups, either a Student’s t-test with a Levene test for homogeneity of variances in the case of normal distribution, or the nonparametric U-Mann Whitney test in the case of non-normal distributions, was applied. PASW18 software was used for statistical analysis. Statistical analysis of the microarray data was performed with the Multi-Experiment p21WAF1/Cip1 Overexpression in a SdhD Mouse Mutant 7 p21WAF1/Cip1 Overexpression in a SdhD Mouse Mutant down-regulated.Date are expressed as the log ratio 6 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. Glut1: 18753409 glucosyltransferase 1, HK2: Hexokinase 2, LDHA: Lactate dehydrogenase A, PDK1: Pyruvate dehydrogenase kinase 1, Vegf: Vascular endothelial growth factor. doi:10.1371/journal.pone.0085528.t001 revealing a non-statistically significant trend towards an increase in heterozygous animals compared with wild-t
