Comparison of gene expression by microarray analysis and qPCR analysis.

<p>Comparison of mRNA expression levels of atrial natriuretic peptide (Nppa), potassium voltagegated channel, Isk-related family, member 1 (Kcne1), EF-hand calcium binding domain 6 (Efcab6) and epoxide hydrolase 2 (Ephx2) at day 20 of fetal development (E20) and week 14 of postnatal life between normotensive strains F344 (white bars) or WKY (grey bars) in comparison to SHRSP (black bars). Data for expression analysis by microarray are shown in the left group, for qPCR analysis in the middle and right groups, respectively. Values are given in % of corresponding control strain F344 or WKY at week 14. *, p < 0.05; **, p < 0.01.</p

Fold changes (logFC) and adjusted p-values of candidate genes in SHRSP and F344.

<p>Fold changes (logFC) and adjusted p-values of candidate genes in SHRSP and F344.</p

Mean systolic blood pressure and relative heart weight.

<p>Comparison of mean systolic blood pressure (panel A) and relative heart weight (panel B) between F344 strain (white bars, E20 n = 8, week 14 n = 8), WKY strain (grey bars, E20 n = 5, week 14 n = 17) and SHRSP (black bars, E20 n = 7, week 14 n = 1) at state E20 and at week 14, respectively. Values are means ± SD, * p < 0.05 vs. F344 at week 14, ** p < 0.05 vs. F344 and WKY at week 14, respectively.</p

Venn diagram of valid and differentially expressed transcripts.

<p>Venn diagram for the candidate identification process to identify genes for further qPCR validation. Significant differential regulation was given with log fold changes > 1.5 or < -1.5 and p < 0.05. First comparison was F344 vs. SHRSP in week 14, second comparison was E20 vs. week 14 in F344 and SHRSP. Validated candidate genes are labelled with asterisk.</p

Body weight and length under standard and cholesterol-containing diets.

<p>Body weights and length of the different genotypes were determined after 184 ± 3 (range 172–194) days after birth. Animals were kept on standard chow or semisynthetic diet containing 0.02% cholesterol. Data are given as means ± SD. Number of animals are given in parenthesis, and results between groups were tested for significance to the respective <i>Mc4r</i>^<i>+/+</i>: *p < 0.05, ** p < 0.01; *** p < 0.001.</p

Correlation between plasma cholesterol levels and plaque sizes of atherosclerotic lesions at the aortic root and brachiocephalic artery (BCA).

<p>Correlations were performed with the data of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167888#pone.0167888.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167888#pone.0167888.g003" target="_blank">Fig 3</a>. To illustrate the results of the multiple regression analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167888#sec002" target="_blank">Material and Methods</a>), we plotted the cholesterol levels against the plaque size for males and females. Furthermore, we added a linear regression line computed separately for <i>Ldlr</i>^<i>-/-</i> with and without <i>Mc4r</i>^<i>mut</i> (regression coefficients are given in the figure, the respective p-values can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167888#pone.0167888.s003" target="_blank">S2 Table</a>).</p

Severe hypercholesterolemia and hypertriglyceridemia in mice lacking both MC4 and LDL receptors.

<p>Serum cholesterol (<b>A</b>) and serum triglycerides (<b>B</b>) were determined in the indicated mouse strains (n = 10–14 male mice). Female mice show essentially similar results (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167888#pone.0167888.t002" target="_blank">Table 2</a>). The statistic evaluation is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167888#pone.0167888.t002" target="_blank">Table 2</a>.</p

Blood glucose, serum urea levels, and liver enzymes on standard chow and semisynthetic diets.

<p>Enzyme activities of the different genotypes were determined after 184 ± 3 days after birth. Animals were kept on standard chow and semisynthetic diet. Data are given as means ± SD and tested for significance to the respective <i>Mc4r</i>^+/+: * p < 0.05, ** p < 0.01; *** p < 0.001. The number of animals/group is between 10 and 14.</p

Quantification of atherosclerotic lesions at the aortic root and brachiocephalic artery (BCA).

<p>Male and female mice (wt: +/+, <i>Mc4r</i> deficiency: mut), all on an <i>Ldlr</i>-deficient background, were fed with standard chow and semisynthetic diet. Atherosclerosis quantification in the BCA (A) and the aortic root (B) was performed after 6 months as previously described (14). Data of the indicated genotypes are given as a scatter and box plot showing the median (bold line) and the lower and the upper quartile (as box) (7–15 animals per group; from left to right: n = 11, 12, 11, 13, 13, 12, 15, 12, 10, 11, 7, 12, 13, 8, 14, 11).</p

Plasma lipids and lipoproteins and liver cholesterol on standard chow and semisynthetic diets.

<p>Plasma lipids and liver cholesterol content of the different genotypes were determined after 184 ± 3 days after birth. Animals were kept on standard chow and semisynthetic diet. Data are given as means ± SD and tested for significance to the respective <i>Mc4r</i>^+/+: *p < 0.05, ** p < 0.01; *** p < 0.001. The number of animals per group was between 10 and 14 (plasma lipids) and 5 (liver cholesterol). n.d., not determined.</p