Fold changes and significance values of normalized transcript levels in mucosal samples of ileal controls (n = 17) when compared to colonic controls (n = 20).

<p>Fold changes and significance values of normalized transcript levels in mucosal samples of ileal controls (n = 17) when compared to colonic controls (n = 20).</p

Tunicamycin induces ER stress in mucosal samples of healthy controls.

<p>Stimulation of paired colonic and ileal samples of five healthy controls with the ER stress inducer tunicamycin revealed an increased expression of <i>HSPA5</i> mRNA in both colonic and ileal tissue, with a more pronounced induction in the ileal mucosa (*P = 0.048). Bars represent the fold induction of <i>HSPA5</i> in treated samples relative to untreated samples.</p

Western Blot analysis of UPR-related proteins.

<p>Immunoblotting was performed on <b>A.</b> colonic and <b>B.</b> ileal samples of 5 healthy controls (HC), 5 active ulcerative colitis (UC) and 5 active Crohn's disease (CD) patients. Signals of HSPA5 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025589#pone-0025589-g003" target="_blank">Fig. 3C</a>), PDIA4 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025589#pone-0025589-g003" target="_blank">Fig. 3D</a>), XBP1s (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025589#pone-0025589-g003" target="_blank">Fig. 3E</a>), GADD34 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025589#pone-0025589-g003" target="_blank">Fig. 3F</a>), and pEIF2A/EIF2A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025589#pone-0025589-g003" target="_blank">Fig. 3G</a>), were quantified. In colonic samples, significant increased concentrations of PDIA4 and pEIF2A/EIF2A were observed in both UC and CD patients, while a significant increase in HSPA5 was only found in UC patients. In ileal samples, no differential expression of the UPR-related proteins was observed. Levels in healthy controls were arbitrary set as 1, and UC and CD levels expressed as the ratio to healthy controls (*P<0.05, **P<0.01).</p

ER stress is mainly localized in epithelial secretory cells.

<p>Immunohistochemical analysis of HSPA5, a central mediator of ER stress, was performed on paraffin embedded slides of <b>A.</b> colonic controls, <b>B.</b> inflamed areas of colonic Crohn's disease (CD) patients, <b>C.</b> inflamed areas of ulcerative colitis patients, <b>D.</b> ileal controls and <b>E.</b> inflamed areas of ileal CD patients. The ER stress observed is linked to the epithelium rather than to recruited inflammatory cells.</p

Transcriptional analysis of UPR-related genes.

<p>Transcript levels of <b>A.... </b><i>HSPA5</i>, <b>B.... </b><i>PDIA4</i>, <b>C.... </b><i>XBP1s∶(XBP1s+XBP1u)</i>, and <b>D.... </b><i>GADD34</i> imply the activation of the ATF6 and IRE1 pathway in inflamed samples of ulcerative colitis (UC) and colonic Crohn's disease (CD) patients, while no differential expression was seen in ileal CD patients when compared to healthy controls. Levels in healthy controls (HC) were arbitrary set as 1, and UC and CD levels expressed as the ratio to healthy controls (*P<0.05, **P<0.01, ***P<0.001).</p

Sequences of qRT-PCR primers, amplicon lengths (bp), PCR efficiencies (%) and correlation coefficients(R²).

<p>Sequences of qRT-PCR primers, amplicon lengths (bp), PCR efficiencies (%) and correlation coefficients(R²).</p

Histology of the joints in WT and TNF^ΔARE mice.

<p>H&E staining of the ankle joints of <b>(A)</b> air-exposed WT mice, <b>(B)</b> CS-exposed WT mice, <b>(C)</b> air-exposed TNF^ΔARE mice and <b>(D)</b> CS-exposed TNF^ΔARE mice. <b>(E)</b> Histological score of inflammation in the joints of WT and TNF^ΔARE mice. Histological inflammation is increased in TNF^ΔARE mice compared to WT mice (p < 0,005). Data are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Inflammatory gene expression in lung tissue and BAL fluid of WT and TNF^ΔARE mice.

<p>mRNA expression of <b>(A)</b> Tnf-α, <b>(C)</b> Tnfr1, <b>(D)</b> Tnfr2, <b>(E)</b> Ccl2, <b>(F)</b> Cxcl1 and <b>(G)</b> Cxcl2 in lung tissue of WT and TNF^ΔARE mice exposed to air or CS during 2 weeks. <b>(B)</b> Protein levels of TNF-α in BAL fluid of WT and TNF^ΔARE mice exposed to air or CS during 2 weeks. mRNA expression of <b>(H)</b> Tnf-α, <b>(J)</b> Tnfr1, <b>(K)</b> Tnfr2, <b>(L)</b> Ccl2, <b>(M)</b> Cxcl1 and <b>(N)</b> Cxcl2 in lung tissue of WT and TNF^ΔARE mice exposed to air or CS during 4 weeks. <b>(I)</b> Protein levels of TNF-α in BAL fluid of WT and TNF^ΔARE mice exposed to air or CS during 4 weeks. Expression levels are relative to the expression of 3 reference genes (hypoxanthine phosphoribosyltransferase 1 (Hprt1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferring receptor protein 1 (Tfrc)). Values are expressed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.</p

Mean clinical arthritis score in WT and TNF^ΔARE mice.

<p>Clinical arthritis score during the course of <b>(A)</b> 2 weeks (WT vs. TNF^ΔARE: p < 0,005) and <b>(B)</b> 4 weeks CS exposure (WT vs. TNF^ΔARE: p < 0,005).</p

Mean TNF-α levels in serum of WT and TNF^ΔARE mice.

<p>Protein levels of TNF-α in serum of WT and TNF^ΔARE mice throughout the 4 weeks air- or CS-exposure. As expected, TNF-α was increased in TNF^ΔARE mice compared to WT mice (p < 0,005).</p