Altered IL-6 and IP-10 levels in SLE patients and lupus relatives.

<p>Plasma levels of IL-6 (<b>A, C</b>) and IP-10 (<b>B, D</b>) were assessed in SLE patients (case), unaffected first-degree relatives of SLE patients (FDR), and unrelated, unaffected controls with no family history of SLE (Ctl). IL-6 (<b>C</b>) and IP-10 (<b>D</b>) levels are compared by genotype of associated SNPs rs76162067 and rs79711023, respectively. Data are presented as mean <u>+</u> SEM. *<i>p<0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i>, ***<i>p<0</i>.<i>001</i>, ****<i>p<0</i>.<i>0001</i> by Kruskal-Wallis test with Dunn’s multiple comparison for all groups assessed (Case/FDR/Ctl [<b>A-B</b>]; Case/FDR/Ctl carrying major (CC) or minor (TC) alleles [<b>C-D</b>]). Only comparisons found to be statistically significant were marked.</p

Genotypic Associations between <i>IFIH1</i> and Immune Mediators.

<p>Zoom plots of associations between SNPs in <i>IFIH1</i> and box-cox transformed plasma levels of (<b>A</b>) IL-6, (<b>B</b>) TNF-α, (<b>C</b>) IFN-β, (<b>D</b>) IP-10, (<b>E</b>) anti-dsDNA, and (<b>F</b>) total number of positive lupus-associated autoantibody specificities (dsDNA, chromatin, Ro/SSA, La/SSB, Sm, SmRNP, and nRNP, as described in <i>Materials and Methods</i>), displayed as negative log transformed p-values (y-axis). Red dots indicate SNPs within Exon 13. LD map of <i>IFIH1</i> SNPs is presented (<b>G</b>).</p

IFIH1 effect on plasma levels of IL-6 and IP-10.

<p>Exon map of IFIH1 is presented (<b>A</b>) containing labeled SNPs rs76162067 and rs79711023. IL-6 (<b>B</b>) and IP-10 (<b>C</b>) levels in study participants expressing the major (CC) or minor (CT) allele of SNP rs76162067 or rs79711023, respectively, are expressed as box-cox transformed (left y-axis) and concentration (right y-axis) values. Exon order is displayed L-R for ease of interpretation, although IFIH1 is transcribed R-L from the negative strand. Data in <b>B-C</b> are presented as median ± interquartile range. *<i>p≤0</i>.<i>05</i>, **<i>p<0</i>.<i>01</i> Mann-Whitney test.</p

European-American Study Participants.

<p>European-American Study Participants.</p

Secondary Structure of Predicted MiRNAs

<p>Predicted hairpin secondary structure of miRNA MIR156a from rice and the newly discovered ortholog from sorghum. The 21-nucleotide MIR156a sequence is highlighted in red.</p

Annotation of <i>Arabidopsis</i> by Sorghum MF Versus Rice Gene Sequences

<p>Shown are the number of <i>Arabidopsis</i> proteins that are matched in a TBLASTN comparison to the sorghum MF set (blue) versus the rice gene sequences (yellow). The <i>Arabidopsis</i> proteins, after having known repetitive elements removed (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030013#s3" target="_blank">Materials and Methods</a>), have been categorized as either hypothetical or known based on the definition line. <i>Arabidopsis</i> proteins were considered supported if they matched with an E-value less than or equal to 1 × 10⁻⁸. Sb, S. bicolor MF set; Osj:seq, <i>Oryza sativa japonica</i> gene sequences.</p

Genome Reduction

<p>MF reduces the sorghum genome by 66% in sampling a hypomethylated space of approximately 247 Mb (green) and filtering out 488 Mb (red) of the 735-Mb sorghum genome.</p

Gene Discovery Rate

<p>Gene discovery rates for sorghum MF (blue), sorghum ESTs (pink), and an <i>Arabidopsis</i> simulation (dotted black) are shown. The gene discovery rates for the MF and ESTs were calculated based on matches to a set of 137 genes annotated on sorghum BAC clones versus the number of MF and EST reads. The <i>Arabidopsis</i> simulation was calculated based on the fold-coverage of chromosome 1, which contains 7,520 genes. The fold coverage was converted into read numbers as detailed in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030013#s3" target="_blank">Materials and Methods</a>.</p

Phylogenetic Comparison of Sorghum <i>DREB1</i> Genes

<p>A phylogenetic tree comparing the AP2 domain of the sorghum <i>DREB1</i> genes to those of <i>Arabidopsis</i> and rice was constructed using CLUSTALX [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030013#pbio-0030013-b61" target="_blank">61</a>]. The genes encoding proteins from <i>Arabidopsis</i> are <i>DREB1A, DREB1B,</i> and <i>DREB1C</i>. Rice genes are <i>OsDREB1A, OsDREB1B, OsDREB1C</i> (nucleotides 142,337–142,981), and <i>OsDREB1D</i> (nucleotides 1,489–2,250). AP2 domains from other <i>Arabidopsis</i> proteins are also included: <i>APETALA2</i> (R2 domain), <i>AtERF-1, LEAFY PETIOLE,</i> and <i>TINY</i>.</p

Methylation Status of <i>tb2</i> and Kafirin Cluster

<div><p>(A and B) Restriction maps of the <i>tb2</i> gene (A) and the kafirin consensus sequences (B) are shown. The relevant restriction sites are indicated vertically and the numbers indicate the distances scale in basepairs. Each CDS is depicted as a blue-shaded arrow, and the region assayed is indicated by a black bar. The circles depict sites that are not present in every kafirin gene, and the color represents the number of genes that do not share the site. The orange circle (5′-most HhaI site) is a site conserved in nine of 11 kafirin genes, and the red circle (3′-most PstI site) is a site present in ten of the 11.</p> <p>(C) Results from a representative methylation analysis of <i>tb2;</i> the inset depicts the template dilution standard curve used to set the threshold for the experiment. Each experiment was performed three times with four on-board replicates per assay point. The results for each of the four differentially treated reactions are depicted with different colors. Red, mock-treated; blue, mcrBC-digested; orange, HhaI-digested; and green, HhaI + mcrBC double-digest. The inset shows the standard dilution control with two replicates at each dilution. The control was used to set the threshold for detection. The specificity of each reaction was confirmed using melt-curve analysis.</p> <p>(D) Results from a representative methylation analysis of the 11 kafirin genes. The results for each of the six differentially treated reactions are the same as in (C), with the following additional digests: pink, PstI-digested; light blue, PstI + mcrBC double-digest. Notice that the mcrBC with and without PstI yields the same Ct, while HhaI + mcrBC (green) yields a higher Ct on average; suggesting additional cleavage.</p></div