Cytochrome <i>c</i> release is inhibited by insulin administration.

<p>(A) Sham operated control animals (Sham, n = 3) show faint detectable amounts of Cyt<i>c</i> in the cytosolic fraction of hippocampal CA1 neurons. After 8 min of ischemia followed by 24 h of reperfusion (R24, n = 5) cytosolic Cyt<i>c</i> increases ∼4 fold (p < 0.05) and there is a trend toward mitochondrial Cyt<i>c</i> decrease (p = 0.08), both indicative of mitochondrial Cyt<i>c</i> release. Animals exposed to 8 min of ischemia followed by 24 h of reperfusion with a single bolus of IV insulin at the onset of reperfusion (T24, n = 5) demonstrate a reduction of cytosolic Cyt<i>c</i> (p < 0.05) and increased mitochondrial Cyt<i>c</i> compared to the R24 controls. (B and C) Densitometric analyses of Western blot data (Mean +/−SEM, *p < 0.05).</p

In vivo induction of cytochrome <i>c</i> Tyr97 phosphorylation in rat brain by insulin treatment.

<p>(A) Cyt<i>c</i> was isolated from rat brain after global brain ischemia (lane 1), from untreated control rats (lane 2), from sham-operated animals after insulin treatment (lane 3), and from rat brain after ischemia with insulin treatment (lane 4). Top, Western blot with an anti-phosphotyrosine antibody (4G10) reveals no detectable tyrosine phosphorylation of brain Cyt<i>c</i> under control conditions and after global brain ischemia (lanes 2 and 1, respectively), whereas tyrosine phosphorylation of Cyt<i>c</i> is strongly induced after insulin treatment (lane 3), but slightly reduced by ischemic stress (lane 4). Bottom, Coomassie gel shows equal loading (1 µg per lane) and purity of the isolated Cyt<i>c</i> species. (B) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after insulin treatment (corresponding to lane 3 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y5, and y6. The sequence of the peptide was definitively assigned by b3, b4, y2, y3, y4, y5, and y6. (C) Nano-LC/ESI-MS/MS analysis of rat brain Cyt<i>c</i> after global brain ischemia and insulin treatment (corresponding to lane 4 in Fig. 5A) unambiguously identifies Tyr97 phosphorylation by fragment ions y3, y4, y6, and y7. The sequence of the peptide was definitively assigned by b3, b4, y3, y4, y6, and y7.</p

Insulin prevents neuronal cell death in the CA1 hippocampus following brain ischemia.

<p>Cresyl violet stained sections (top row) show CA1 hippocampus densely populated with pyramidal neurons in sham-operated controls (Sham), and triple-label immunofluorescence (bottom row) shows these cells to be NeuN-positive (red). After 8 min of global brain ischemia followed by 14 days of reperfusion (I/R, n = 5) there is a 90% loss of CA1 neurons and an increase in Iba-1-positive microglia and GFAP-positive astrocytes (green and magenta, respectively). Animals exposed to 8 min of ischemia followed by 14 days of reperfusion with a single bolus of insulin given at the onset of reperfusion (I/R + Ins, n = 4) demonstrate a 49% increase in NeuN-positive neurons (p < 0.05) while Iba-1 and GFAP-positive cells remain unchanged.</p

Nano-LC/ESI-MS/MS spectrum of EDLIApYLKKATNE.

<p>Peptides were eluted into the mass spectrometer by applying a HPLC gradient of 0–70% 0.1 M acetic acid/acetonitrile in 30 minutes. The mass spectrometer acquired top 9 data dependent ESI MS/MS spectra. The phosphorylation site was unequivocally assigned by fragment ions b6, b8, b9, b10, and y3, y4, y6. The sequence of the peptide was definitively assigned by b3, b6, b8, b9, b10, b11, b12, and y2, y3, y4, y6, y9, y10, y11.</p

Insulin treatment leads to phosphorylation of brain cytochrome <i>c</i>.

<p>Ischemic pig brain tissue was treated +/− insulin following Cyt<i>c</i> purification under conditions that preserve protein phosphorylation. (A) Western analysis with an anti-phosphotyrosine antibody (4G10) indicates tyrosine phosphorylation of Cyt<i>c</i> after insulin treatment (lane 4), whereas Cyt<i>c</i> isolated without insulin treatment (lane 5) or obtained from a commercial source (lane 3, Sigma Cyt<i>c</i>) does not produce any signal. Lane M, protein size marker (kDa); lane 1, EGF stimulated A431 cell lysate (positive control for Western analysis); lane 2, ovalbumin (negative control for Western analysis). Western analysis with anti-phosphoserine and anti-phosphothreonine antibodies did not show any signal (not shown). (B) Control Western blot with an anti-Cyt<i>c</i> antibody shows similar loading. Samples in lanes 1–5 as denoted in A. Note that additional bands in Sigma Cyt<i>c</i> correspond to the Cyt<i>c</i> dimer and trimer that are sometimes observed depending on the batch.</p

Criteria for selection of mutant mouse line.

<p>^apublished data</p><p>^boriginal data from GMC screen</p><p>Criteria for selection of mutant mouse line.</p

Lung transcriptome comparison of mutant versus wild type animals following OVA challenge.

<p>(A) Summarized heat map of genes regulated after OVA challenge in all three mutant mouse lines compared to the respective challenged control littermates. Genes with similar expression patterns are grouped together (A to G). Color code indicates the mean fold changes of the respective genes in one group for each OVA challenged mutant mouse compared to the mean of the corresponding challenged wild type littermates. Orange represents up- and blue down-regulation in the mutant mice. The column “# genes” displays the number of genes that are included in each gene group A to G (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134503#pone.0134503.s003" target="_blank">S2D Fig</a> shows the corresponding data for individual genes). (B) Venn diagram representing the overlap of gene regulation. (C) Summarized heat map of overlapping gene expression in at least 2 of the 3 mutant mouse lines. Grey boxes indicate that there is no significant regulation of these genes in the respective mutant mouse lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134503#pone.0134503.s003" target="_blank">S2E Fig</a> shows the data for individual genes of this panel).</p

Immunoglobulin isotype levels in mutant mice before and after OVA challenge.

<p><b>(</b>A) <i>Cox4i2</i>^{<i>tm1Hutt</i>}, (B) <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and (C) <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>}. Box plots show levels of immunoglobulin isotypes in the respective mutant mice and corresponding wild type littermates after OVA challenge. Grey boxes display the interquartile range of each immunoglobulin isotype under standard conditions (+/+: wild type,-/-: respective mutant mice; p-value * < 0.05; n = 10–12 per group).</p

Comparative analysis of transcriptome data under standard conditions.

<p>(A) Summarized heat map from HCL analysis of genes regulated in the three mutant mouse lines. Genes with similar expression patterns are grouped together (A to E). Color code indicates the mean fold change of the respective genes in one group for each mutant mouse compared to the mean of the corresponding wild type littermate group. Orange represents up- and blue down-regulation in the respective mutant mice. The column “# genes” gives the number of genes that are included in each group A to E. (B) Venn diagram of differentially expressed genes common or specific in <i>Cox4i2</i>^{<i>tm1Hutt</i>}, <i>Ifit2</i>^{<i>tm1</i>.<i>1Ebsb</i>} and <i>Prdm11</i>^{<i>tm1</i>.<i>1ahl</i>} mutant mice.</p

Total IgE levels in plasma before and after OVA challenge.

<p>Box plots show total IgE levels after OVA challenge in mutant mouse lines and corresponding wild type littermates. Grey boxes show the interquartile ranges of total IgE levels under standard conditions of the respective groups (p-value ** < 0.01, n = 11–31 per group).</p