Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.</p

Differences in gene expression between WT and FTO-4 mice based on microarray data.

<p>Expression of the indicated genes in the cerebellum (A), hypothalamus (B), WAT (C) and gastrocnemius (D) of wildtype (WT, black bars) and FTO-4 (white bars) mice. Data are expressed as the fold change compared to wildtype. Significance was tested using Student's t-test to compare FTO-4 to WT. ***p<0.001, **p<0.01, *p<0.05.</p

Effect of FTO overexpression on m6A levels in RNA.

<p>FTO mRNA expression in wild-type (black bar) and FTO-4 (white bar) MEFs as the fold change compared to wild-type (A). m6A levels measured as a percentage of adenosine levels for mRNA and total RNA by LC/MS in wild-type (black bar) and FTO-4 (white bar) MEFs (B). Significance was tested using Student's t-tests to compare FTO-4 to WT data. **p<0.01.</p

Overview of potential m6A sites in obesity-related genes with changed expression in FTO-4 mice.

<p>Pie chart of obesity-related genes whose expression is changed in the indicated tissues of FTO-4 mice. Dark grey, m6A sites in their mRNA transcripts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Meyer1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Dominissini1" target="_blank">[24]</a>. Light grey, no m6A sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Meyer1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone.0097162-Dominissini1" target="_blank">[24]</a>. The number of genes involved is indicated: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097162#pone-0097162-t002" target="_blank">Table 2</a> for gene names.</p

Top 10 genes that showed the highest amount of overexpression in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of overexpression in FTO-4 mice per tissue.</p

qPCR analysis of the effect of m6A on DNA amplification.

<p>qPCR analysis using different concentrations of the DNA templates ACTB (A), HPRT1 (B) and HSPA8 (C) containing m6A (open squares) or unmethylated adenosine (black circles) revealed that m6A does not interfere with DNA amplification.</p

Glycolysis upregulation upon <i>Candida</i> stimulation.

<p>(A) Schematic pathway map of the gene expression in the main metabolic pathways in PBMCs stimulated with heat-killed <i>C</i>. <i>albicans</i> yeast 24 h after stimulation. The dots represent metabolites, and the arrows indicate reactions converting these metabolites. For each reaction it is known which enzymes (and thus which genes) are involved in catalyzing the reaction. The arrows marked in red indicate an overall upregulation of genes involved in those reactions in <i>C</i>. <i>albicans</i> versus RPMI, whereas the blue indicates a downregulation. A darker color indicates a larger change in transcript levels. The complete map stimulation as created by Escher for 4 h and 24 h stimulation is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006632#ppat.1006632.s001" target="_blank">S1 Fig</a>. (B) Fold increase of mRNA expression for the indicated enzymes analyzed by RT-PCR in monocytes 24 h after stimulation with heat-killed <i>C</i>. <i>albicans</i> conidia or heat-killed <i>C</i>. <i>albicans</i> hyphae (mean ± SEM, n = 6–9; pooled from 2–3 experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. HK2: Hexokinase 2; PFKP: Phosphofructokinase, platelet; α-KGDH: alpha-ketoglutarate dehydrogenase; LDH: Lactate dehydrogenase; mTOR: Mammalian target of rapamycin; GLS: Glutaminase; GLUD: Glutamine dehydrogenase.</p

Inhibition of glucose metabolism impaired <i>in vivo</i> responses to systemic <i>C</i>. <i>albicans</i> infection.

<p>(A) Fungal burden measured in the kidneys of C57BL/6 mice treated with PBS, 2-DG or BPTES during 5 days after i.v. <i>C</i>. <i>albicans</i> challenge (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments). *p<0.05, Student’s t test. Each dot represents one mouse. (B) Candidacidal activity of neutrophils isolated from blood of C57BL/6 mice treated with PBS, 2-DG or BPTES during 5 days after i.v. <i>C</i>. <i>albicans</i> challenge (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments) *p < 0.05, Student’s t test. Each dot represents one mouse. (C) IL-1β, IL-6, IL-10, IFNγ and TNFα production by mouse splenocytes obtained from PBS, 2-DG or BPTES-treated mice 5 days after <i>C</i>. <i>albicans</i> i.v. infection were measured by ELISA 48 h after <i>in vitro</i> restimulation with medium, LPS, heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae (mean ± SEM, n = 6; similar results were obtained in 2 independent experiments) *p<0.05, Student’s t test.</p

<i>Candida</i> stimulation induced glycolysis in human monocytes.

<p>(A) Lactate production and glucose consumption by monocytes after overnight stimulation with heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae (mean ± SEM, n = 12 for lactate, n = 6 for glucose; pooled from 2–4 independent experiments). *p<0.05, ***p<0.001 Wilcoxon signed-rank test. (B-D) Basal and maximum extracellular acidification rates (ECAR; B), basal and maximum oxygen consumption rates (OCR; C) or spare respiratory capacity (SRC; D) of monocytes were determined by Seahorse analysis at 4 and 24 h after stimulation with medium or heat-killed <i>C</i>. <i>albicans</i> yeast (mean ± SEM, n = 6–8; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. (E) Intracellular metabolite levels of monocytes 4 and 24 h after heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae stimulation (mean ± SEM, n = 6–8; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test. (F) Lactate production by monocytes after overnight stimulation with live <i>hgc1</i> or <i>Δhgc1 C</i>. <i>albicans</i>. (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, Wilcoxon signed-rank test. (G) Intracellular metabolite levels of monocytes 4 and 24 h after <i>hgc1</i> or <i>Δhgc1</i> live <i>C</i>. <i>albicans</i> stimulation (mean ± SEM, n = 6; pooled from 2 independent experiments). *p<0.05, **p<0.01 Wilcoxon signed-rank test.</p

ROS production by monocytes involved glycolysis and the pentose phosphate pathway.

<p>(A-D) Human monocytes were treated with medium (A), 2-DG (B), DCA (C) or 6-AN (D) and subsequently stimulated with medium, heat-killed <i>C</i>. <i>albicans</i> yeast or heat-killed <i>C</i>. <i>albicans</i> hyphae. Luminescence generated from ROS production was measured every 145 seconds during 60 minutes (n = 4; pooled from 2 independent experiments). Dashed lines show maximum ROS levels reached without inhibitor treatment to be used as a visual reference.</p