mRNA microarray expression analysis in animals after two weeks of stress (2WSTRESS), recovery from stress (4WSTRESS) and appropriate controls for each time point (2WCONTROL, 4WCONTROL).

<p>Genes with a 2-fold difference and a p-value of p<0.05 are shown in logarithmic scale (log₂). Genes that were changed are represented as dark-red diamonds, in top-left (down-regulated) and top-right (up-regulated) parts of each figure. 2S_2C – groups of 2WSTRESS vs. 2WCONTROL, 4C_2C – 4WCONTROL vs. 2WCONROL, 4S_2S – 4WSTRESS vs. 2WSTRESS, 4S_2C – 4WSTRESS vs. 2WCONTROL.</p

sq-RT-PCR analysis.

<p>A: prolactin receptor (<i>Prlr</i>) gene; B: <i>Adipoq</i> gene. C: ephrin B3 receptor (<i>Efnb3</i>) gene; D: GABA (A) receptor 4 (<i>Gabra4</i>) gene. E: GAPDH. Data are represented as an average of three animals per group. Asterisks represent statistical significance (* p<0.05; ** p<0.01; *** p<0.001). Error bars represent standard deviation of the mean. Photographs below bars represent corresponding PCR fragments in duplicates for each animal for three animals per group.</p

A: Dose-dependent inhibition of <i>Eps15</i> expression in the Luciferase Assay after transfection of HEK-293 cells with miR-186.

<p>B: Dose-dependent inhibition of the <i>Nab1</i> gene in the Luciferase Assay after transfection of MCF7 cells with miR-709. C: Luciferase Assay with pFN4 (3′UTR <i>Eps15</i>) and miR-186. The first bar demonstrates relative level of luciferase activity after transfection of MCF-7 cells with pFN4 only. Second bar: pFN4 + miR-186. Third bar: pFN4 mut + miR-186. Asterisks represent statistical significance (p<0.001). D: Luciferase Assay with pFN7 (3′UTR <i>Nab1</i>) and miR-709. The first bar demonstrates relative level of luciferase activity after transfection of MCF-7 cells with pFN7 only. Second bar: pFN7 + miR-709. Third bar: pFN7 mut + miR-709. Bars represent the normalized average of relative luciferase units.</p

Skilled reaching performance in male rats.

<p>A, B: Series of photographs illustrating forelimb and digit movements of a rat grasping a food pellet. C: skilled reaching success in rats before (baseline), during and after two weeks of stress (mean ± SEM). Note that stress diminished skilled reaching performance after acute (day 1) and chronic (day 14) of daily stress treatment. Reaching success did not return to baseline levels within 14 days of recovery. Asterisks represent statistical significance (*** p<0.001).</p

List of genes changed after two weeks of restraint treatment (2WSTRESS vs. 2WCONTROL).

<p>List of genes changed after two weeks of restraint treatment (2WSTRESS vs. 2WCONTROL).</p

qRT-PCR analysis of miR-186 (A,C,E) and miR-709 (B,D,F) expression level in cerebellum (A,B), hippocampus (C,D), and prefrontal cortex (E,F).

<p>Data are represented as a normalized relative fold change to control. Asterisks represent statistical significance (*p<0.1; **p<0.05; ***p<0.001). Control animals are represented in black, stressed animals in grey. For more details see (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029441#pone.0029441.s013" target="_blank">Tables S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029441#pone.0029441.s014" target="_blank">S8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029441#pone.0029441.s015" target="_blank">S9</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029441#pone.0029441.s016" target="_blank">S10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029441#pone.0029441.s017" target="_blank">S11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029441#pone.0029441.s018" target="_blank">S12</a>).</p

Comparison of gene expression levels between groups undergoing 2 weeks of stress (2WS) and controls (2WC) and recovery from stress (4WS) and controls (4WC).

<p>Comparison of gene expression levels between groups undergoing 2 weeks of stress (2WS) and controls (2WC) and recovery from stress (4WS) and controls (4WC).</p

Concentration of plasma corticosterone (means ± SD; ng/ml) in control and stress animals as measured on the first day of stress, last day of stress and after two weeks of recovery from stress (“Recovery”).

<p>Asterisks represent statistical significance (*** p<0.001).</p

Functional annotation clustering.

<p>Green - corresponding gene-term association positively reported; black - corresponding gene-term association not reported yet. A: cluster of four genes: <i>Tcf21</i>, <i>Msx1</i>, <i>Adipoq</i>, <i>Cited</i>, which were grouped based on their involvement in positive regulation of the macromolecule metabolic process. B: cluster of three genes: <i>Prlr</i>, <i>Otc</i>, <i>Adipoq</i>, which were grouped based on their involvement in protein complex assembly. C: cluster of four receptors: <i>Prlr</i>, <i>Osmr</i>, <i>Itgb6</i>, <i>Ssta6</i>. D: cluster of three genes: <i>Cldn3</i>, <i>Itgb6</i>, <i>Cdh3</i>, which were grouped based on their involvement in cell adhesion.</p

Analysis of miRNA expression in the rat cerebellum after two weeks of stress compared to controls.

<p>The microarray heatmap demonstrates the log₂ ratio of miRNA signal difference between control and stress samples. Up-regulated miRNAs are shown in red, while down-regulated miRNAs are shown in green. The first three columns on each figure represent the level of expression in control animals, while the three last columns represent the level of corresponding miRNA expression in stress animals.</p