A: Expression of <i>GSTCD</i> and <i>INTS12</i> mRNA in Lung and Airway cells.

<p>mRNA expression in human airway smooth muscle (HASM) cells, human bronchial epithelial cells (HBEC) and peripheral blood mononuclear cells (PBMC) is shown relative to mRNA from lung. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=3). Only the expression of <i>GSTCD</i> in HBEC relative to lung was statistically significant (* <i>P</i>=0.0494). <b>B</b>: <b>Correlation between <i>GSTCD</i> and <i>INTS12</i> ΔCt values in HASM, HBEC, PBMC and lung</b>. mRNA expression levels as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone-0074630-g003" target="_blank">Figure 3A</a> from human airway smooth muscle (HASM) cells, human bronchial epithelial cells (HBEC), peripheral blood mononuclear cells (PBMC) and lung were correlated using a scatter plot. The correlation coefficient between these measures was r=0.8, <i>P</i><0.0001. <b>C</b>: <b>Correlation between <i>GSTCD</i> and <i>INTS12</i> mRNA levels in the lung</b>. The scatter plot shows a positive correlation between the <i>GSTCD</i> and <i>INTS12</i> probe sets as investigated in the lung eQTL study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B20" target="_blank">20</a>].</p

<i>GSTCD</i> and <i>INTS12</i> gene expression is altered following exposure of HASM cells to TGFβ1.

<p>Human airway smooth muscle (HASM) cells were exposed to 10ng/ml TGFβ1 for 4 or 24 hours. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=5). Significant increases in both <i>GSTCD</i> and <i>INTS12</i> gene expression were observed following 24h exposure to TGFβ1 (<i>P</i><0.05 <i>GSTCD</i>, <i>P</i><0.01 <i>INTS12</i>) and after 4h TGFβ1 exposure in <i>INTS12</i> expression (<i>P</i><0.05).</p

Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see Table S5 for patient demographics).

<p>Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone.0074630.s009" target="_blank">Table S5</a> for patient demographics).</p

Regulatory motifs within the <i>GSTCD/INTS12</i> locus.

<p>The <i>GSTCD</i>/<i>INTS12</i> locus is shown, annotated with RNA sequencing, H3K27Ac histone marks, DNase hypersensitivity, transcription factor binding and CpG islands (UCSC Genome Browser (<a href="http://genome.ucsc.edu/" target="_blank"><u>http://genome.ucsc.edu/</u></a>)) on the Human Feb 2009 (GRCh37/hg19) assembly. For the H3K27Ac histone marks and RNA sequence tracks, peak height is proportional to signal amplitude, with colours representing datasets in different cell backgrounds (pale blue H3K27Ac histone trace = human umbilical vein endothelial cell (HUVEC); blue/grey = K562 erythroleukaemia cells). For the DNase hypersensitivity and transcription factor binding tracks, a grey band indicates the extent of the hypersensitive region and the intensity of the band is proportional to the maximum signal strength observed in any cell line.</p

Genetic architecture of the region containing both <i>GSTCD</i> and <i>INTS12</i> genes.

<p>The top panel depicts gene arrangements previously reported in NCBI, build 37, whereas the lower panel shows novel variants identified in lung. V1, 2, 3 refer to splice variants 1, 2 and 3 for each gene. Open boxes represent exons and connecting black lines represent introns. Also illustrated are the locations of Single Nucleotide Polymorphisms (SNPs) meeting genome-wide association (<i>P</i>≤5x10^-8) for FEV₁ in previously reported analyses of the SpiroMeta consortium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B5" target="_blank">5</a>]. Highlighted in red is the sentinel SNP rs10516526 that was associated with FEV₁ (<i>P</i>=2.18 x 10^-23 in all stage analyses) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B5" target="_blank">5</a>]. Translation start codons (ATG) are shown boxed in green and stop codons (TAA) boxed in red.</p

Fetal lung gene array data for <i>AGER</i> expression across Pseudoglandular and Canalicular stages.

<p>Fetal lung gene array data for <i>AGER</i> expression across Pseudoglandular and Canalicular stages.</p

<i>AGER</i> isoform expression in three HBEC donors using RNA Seq.

<p>Structure and abundance of known <i>AGER</i> isoforms in three human bronchial epithelial cell donors illustrating heterogeneity in expression levels. Percentage abundances (% FPKM) were calculated for each donor. Transcripts for full length and soluble <i>AGER</i> were identified at similar low abundancies. FPKM; fragments per kilobase of transcript per million mapped reads.</p

<i>AGER</i> mRNA expression levels in total lung and airway cells.

<p>Q-PCR analysis identified <i>AGER</i> mRNA was highly expressed in total lung and at lower levels in human airway smooth muscle cells (HASM) cells, human bronchial epithelial cells (HBEC) and the BEAS-2BR1 bronchial epithelial cell line, (n = 3).</p

Baseline characteristics of UK smoker population used for genetic association studies.

<p>Baseline characteristics of UK smoker population used for genetic association studies.</p

Immunohistochemical analysis of RAGE expression in healthy and COPD lung.

<p>In healthy lung tissue, RAGE was found to be localised to the cytoplasm and membrane. RAGE expression was high in the pneumocytes of alveolar regions (a). The bronchial epithelium showed variable weak to moderate staining (e). In lung tissue of individuals with COPD, RAGE was very strongly immunopositive in the membrane and cytoplasm of the pneumocytes in the alveolar regions (b). The bronchial epithelium from individuals with COPD was weak or immunonegative for the RAGE protein (f). All isotype controls were negative (c, d, g and h). Representative images of one healthy and one COPD lung shown. x10 magnification.</p