Inhibition of p38 and p44/42 MAPK signaling by siRNA transfection.

<p>OASF (n = 6) were transfected with siRNA targeting p38 MAPK, p44/42 MAPK and scrambled siRNA (control) before ATP (100 µM) stimulation for 2 h. <b>A</b>. Decreased p38 and p44/42 MAPK expression after siRNA transfection was verified by Western blot analysis. <b>B</b>. Densitometric analysis of Western blot results. <b>C</b>. The ATP-induced BDNF mRNA expression was reduced in p38 MAPK siRNA transfected OASF but not in p44/42 MAPK siRNA transfected cells. Data are shown as means ± standard deviations<b>.</b> *, p<0.05.</p

Purinergic receptor P2X, but not P2Y agonists induce BDNF expression.

<p>OASF (n = 8) and RASF (n = 3) were stimulated for 2 h with 100 µM ATP, ADP and UTP, respectively. BDNF mRNA expression was increased by ATP and ADP, but not by UTP. Data are shown as means ± standard deviations. N.s., not significant; *, p<0.05, ***, p<0.001.</p

Activation of MAPK pathways in OASF after ATP stimulation.

<p><b>A</b>. OASF were stimulated with ATP (100 µM) for 10 minutes, 2 h, 5 h and 24 h. Western blot analysis of p38 and p44/42 MAPK, as well as phosphorylated p-p38 and p-p44/42 MAPK showed kinase activation after 10 minutes ATP stimulation. <b>B</b>. OASF (n = 10) and RASF (n = 3) were treated with the kinase inhibitors FR180204, SB203580 and PD98059, respectively 15 min before ATP (100 µM) stimulation for 2 h. Data are shown as means ± standard deviations<b>.</b> *, p<0.05.</p

Additional file 1: of Tie2 as a novel key factor of microangiopathy in systemic sclerosis

Supplemental methods (DOC 43 kb

Additional file 3: Figure S2. of Tie2 as a novel key factor of microangiopathy in systemic sclerosis

Changes of the expression of angiopoietins and Tie2 in chronic hypoxia in vivo. (A) shows no effect of hypoxia on the levels of Ang-1 mRNA, whereas Ang-2 (B) and Tie2 mRNA transcripts (C) increased compared to normoxia. RNA from three mice kept in hypoxia and nine mice kept in normoxia were analysed by qRT-PCR. (TIF 371 kb

Additional file 2: Figure S1. of Tie2 as a novel key factor of microangiopathy in systemic sclerosis

Induction of skin fibrosis in the murine bleomycin skin model. (A) shows the increase in dermal thickness upon bleomycin treatment (HE staining) whereas (B) depicts the increased deposition of extracellular matrix proteins (Sirius Red staining). (C) and (D) show the semi-quantitative analysis of dermal thickness measurements and of hydroxyproline contents. Pictures are representative examples of four bleomycin-treated and four saline-treated controls. (TIF 71 kb

Qualitative results of reverse transcription-polymerase chain reactions (PCRs) using DREAM primer and DREAM nested primer

DREAM amplicons of 409 base pairs (bp) in size in total RNA derived from cerebellum and spinal cord, which served as positive controls. Amplicons of the expected size after reamplification from total RNA isolated from normal synovial fibroblast-like cells (NSFLCs) and osteoarthritis synovial fibroblast-like cells (OA-SFLCSs). DREAM amplicon of 409 bp and the amplicon resulting from nested PCR, starting from the PCR mix, which did not show any product on the agarose gel. The size of the smaller amplicon corresponds to the expected size of 276 bp. Sequence of the amplicon. Positions of primers are highlighted in bold (DREAM forward and reverse) and bold italics (nested DREAM forward and reverse). DREAM, downstream regulatory element antagonist modulator.<p><b>Copyright information:</b></p><p>Taken from "DREAM is reduced in synovial fibroblasts of patients with chronic arthritic pain: is it a suitable target for peripheral pain management?"</p><p>http://arthritis-research.com/content/10/3/R60</p><p>Arthritis Research & Therapy 2008;10(3):R60-R60.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2483451.</p><p></p

Relative DREAM gene expression in peripheral blood mononuclear cells from osteoarthritis (OA) patients and healthy controls

Relative gene expression was normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and is given as delta CT (dCT) value, with higher values representing lower expression levels. DREAM gene expression was significantly lower in OA patients with a high pain score (visual analog scale [VAS] score of greater than 40; △) compared with healthy controls (○) and with OA patients with a low pain score (VAS score of less than or equal to 40; ∇). No significant differences were observed between healthy controls and OA patients with a VAS score of less than or equal to 40. Statistics: one-way analysis of variance followed by Tukey's honest significant difference (*< 0.05). Ctrl, control; DREAM, downstream regulatory element antagonist modulator.<p><b>Copyright information:</b></p><p>Taken from "DREAM is reduced in synovial fibroblasts of patients with chronic arthritic pain: is it a suitable target for peripheral pain management?"</p><p>http://arthritis-research.com/content/10/3/R60</p><p>Arthritis Research & Therapy 2008;10(3):R60-R60.</p><p>Published online 28 May 2008</p><p>PMCID:PMC2483451.</p><p></p

<i>ELMO1</i> promotes cell migration and invasion by RA FLS.

<p>(<b>A</b>) Cell migration in the wound closure assay shows that <i>ELMO1</i> knockdown with siRNA decreases cell migration whereas the control or ELMO2 siRNAs do not. Mean and SEM was calculated from 5 experiments for <i>ELMO1</i> and 3 experiments for ELMO2. (<b>B</b>) Cell invasion assay shows that <i>ELMO1</i> promotes the movement of FLS into extracellular matrix. Levels were calculated in the presence and absence (MED) of PDGF and with control and <i>ELMO1</i> siRNA. Mean cell number and SEM was calculated from 10x field-of view images and then normalized to control. (<b>C</b>) Fields of view showing FLS invading through a Martigel layer. Note the decreased number of invading cells when the FLS are pre-treated with <i>ELMO1</i> siRNA.</p

<i>ELMO1</i> methylation and expression in RA FLS and synovium.

<p>(<b>A</b>) A summary of DNA methylation at the <i>ELMO1</i> promoter regions from our previous study of FLS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124254#pone.0124254.ref004" target="_blank">4</a>] and the location of the <i>ELMO1</i> RA GWAS SNP. The methylation levels of each of the CGs on the bead array that are within the <i>ELMO1</i> promoter region (-2500bps to +500bps from TSS) are shown. Error bars represent the standard error of the mean (SEM) and stars highlight significantly differentially methylated CGs. The locations of the TSSs are indicated with arrows and the transcript variant numbers of the RefSeq genes that are transcribed from that TSS are shown. Comparisons are shown between RA, osteoarthritis (OA) and normal (NL) FLS. (<b>B</b>) Western blots showing the expression of and GAPDH in RA and OA synovial tissue. First lane shows positive control, and subsequent lanes show <i>ELMO1</i> protein levels in whole RA and osteoarthritis (OA) synovium (n = 4 each). There was no significant difference in overall <i>ELMO1</i> expression. (<b>C</b>) Immunohistochemistry for <i>ELMO1</i> expression in RA and OA synovial tissue. Note prominent staining in synovial intimal lining and sublining perivascular regions (brown color). Osteoarthritis synovium had a similar distribution. Left panels shows anti-<i>ELMO1</i> antibody. Right panels shows control IgG. Tissues were lightly counterstained with hematoxlin. Original image at 200x magnification.</p