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

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

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

GSTCD and INTS12 protein expression in human tissue.

<p>Immunohistochemistry studies assessed GSTCD and INTS12 protein expression in tissue sections from controls, individuals with COPD and from a range of fetal developmental stages. All images x40 magnification. <b>A</b>: Representative images of GSTCD expression in lung tissue from three healthy donors (images a and e) with matched isotype controls (iso, images c and g) and in lung tissue from three donors with COPD (images b and f) with matched isotype controls (iso, images d and h). <b>B</b>: Representative images of INTS12 expression in lung tissue from three control donors (images a and e) with matched isotype controls (iso, images c and g) and in lung tissue from three donors with COPD (images b and f) with matched isotype controls (iso, images d and h). <b>C</b>: Panel 1: GSTCD expression in tissue from human fetuses at a range of developmental stages: embryonic (19 days, a and b; 21 days, c and d; 23 days, e and f); pseudoglandular (10 weeks, g and h; 12 weeks, i and j); canalicular (17 weeks, k; 19 weeks, l). Expression was observed to be increased through the pseudoglandular stage. Panel 2: INTS12 expression in the same panel of tissue samples as described above. Isotype controls were all negative (data not shown).</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

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

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