Additional file 1: Figure S1. of A20 suppresses canonical Smad-dependent fibroblast activation: novel function for an endogenous inflammatory modulator

TGF-ß suppresses A20 expression in adult skin fibroblasts. Confluent adult skin fibroblasts were incubated with TGF-ß2 (10 ng/ml) for indicated time-points. A RNA was examined by qPCR. Results, normalized with GAPDH, are means ± SD of triplicate determinations (n = 2). B Western analysis of whole-cell lysates. Representative immunoblots (n = 2). Band intensities, normalized for beta actin in each lane are shown below (PDF 50 kb

Oligonucleotides used for Real-Time Quantitative PCR.

<p>Oligonucleotides used for Real-Time Quantitative PCR.</p

Clinical characteristics of scleroderma patients studied.

<p>M, male; F, female.</p

Concentrations of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the supernatants of spleen cells harvested from mice with collagen-induced arthritis and treated with 1-methyl-tryptophan (1-MT) or vehicle

<p><b>Copyright information:</b></p><p>Taken from "Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice"</p><p>http://arthritis-research.com/content/9/3/R50</p><p>Arthritis Research & Therapy 2007;9(3):R50-R50.</p><p>Published online 18 May 2007</p><p>PMCID:PMC2206348.</p><p></p> The release of IL-4 and IFN-γ into the supernatants of spleen cells in response to human or mouse type II collagen (CII) was determined at the end of the experiment. Values are presented as the mean and standard error of the mean of 16 animals per group (*< 0.01)

TGF-β stimulates Nab2 expression.

<p>Confluent cultures of human foreskin fibroblasts were incubated with TGF-β1 (10 ng/ml) for indicated periods. A, D Total RNA was isolated and analysed by real-time quantitative PCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. B. Whole cell lysates were subjected to Western analysis. Representative immunoblots. C. Fibroblasts were fixed and stained with anti-Nab2 antibodies, or DAPI to detect nuclei, and viewed by immunofluorescence microscopy (original magnification ×100).</p

Increased skin collagen accumulation in Nab2 ^−/− mouse.

<p>Skin from Nab2^−/− mice and wildtype mice was harvested. A. Tissues were stained with hematoxylin and eosin (a–d) or picrosirius red (e,f). Original magnification ×100 (a, b), ×400 (c–f). B. Immunofluorescence using antibodies to α-smooth muscle actin (green). Nuclei were identified by DAPI (blue). Original magnification ×100 (a, d), ×200 (b, c, e, f). C. Total RNA was harvested and was subjected to real-time qPCR analysis. Results, expressed relative to 18S, are the means±S.D. of triplicate determinations from a representative experiment.</p

Nab2 blocks Egr-1-dependent collagen stimulation.

<p>A. Foreskin fibroblasts were cotransfected with expression vector for wildtype or mutant Egr-1 along with Nab2 or empty vector, and 376COL1A2-luc_reporter constructs. Following incubation of cultures for 24 h, cell lysates were prepared and assayed for their luciferase activities. Results, normalized with Renilla luciferase, are expressed as means±S.D. of triplicate determinations. *p<0.005. B. Foreskin fibroblasts were infected with Ad-Egr-1m along with Ad-Nab2. Following 24 h incubation, whole cell lysates were prepared and subjected to Western analysis. Representative immunoblots.</p

Humoral immune responses in DBA mice immunized with type II collagen (CII) and complete Freund's adjuvant (CFA) and treated with 1-methyl-tryptophan (1-MT) or vehicle

<p><b>Copyright information:</b></p><p>Taken from "Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice"</p><p>http://arthritis-research.com/content/9/3/R50</p><p>Arthritis Research & Therapy 2007;9(3):R50-R50.</p><p>Published online 18 May 2007</p><p>PMCID:PMC2206348.</p><p></p> Concentrations of antibodies (total concentrations of IgGAM [immunoglobulins G, A, and M]) to heterologous (human) CII were determined in the serum of DBA/1 mice immunized with CII and CFA and treated with 1-MT or vehicle. Concentrations of antibodies (total concentrations of IgGAM) to autologous (mouse) CII were determined in the serum of DBA/1 mice immunized with CII and CFA and treated with 1-MT or vehicle. Sera were obtained on days 5, 22, 33, and 54. Values are presented as the mean and standard error of the mean of 25 1-MT-treated and 20 vehicle-treated DBA/1 mice per group and represent two independent experiments. *< 0.05 between vehicle and 1-MT-treated mice on the corresponding days

Incidence and severity of collagen-induced arthritis (CIA) in mice treated with 1-methyl-tryptophan (1-MT) or vehicle

<p><b>Copyright information:</b></p><p>Taken from "Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice"</p><p>http://arthritis-research.com/content/9/3/R50</p><p>Arthritis Research & Therapy 2007;9(3):R50-R50.</p><p>Published online 18 May 2007</p><p>PMCID:PMC2206348.</p><p></p> Arthritis was first detected on day 26 after immunization with type II collagen on days 0, 21, and 42 (solid arrows) in mice treated with 1-MT or vehicle (open arrows). Incidence of CIA expressed as the percentage of arthritic animals. Disease severity expressed as the cumulative arthritis score in affected animals. Statistically significant differences in arthritis scores were found between days 37 and 47 (< 0.05). Values are presented as the mean and standard error of the mean of 25 1-MT-treated and 20 vehicle-treated DBA/1 mice per group and represent two independent experiments

Enhanced collagen stimulation in Nab2-deficient fibroblasts.

<p>Wildtype and Nab2^−/− mouse embryonic fibroblasts (MEFs) cultured in parallel were incubated with or without TGF-β1 for 24 h. A. Whole cell lysates and culture supernatants were subjected to Western analysis. Representative immunoblots. B. Total RNA was subjected to real-time qPCR analysis. Results, expressed relative to 18 s, are the means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. C. MEFs were transfected with pEBS₄-luc inpresence or absence of Nab2. Following 24 h incubation, cultures were harvested and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. D. siRNA knockdown. Normal dermal fibroblasts were transfected with Nab2 siRNA or irrelevant negative control siRNA, and incubated with TGF-β. Twenty-four h later, fibroblasts were harvested. Levels of Nab2 and collagen were determined by Western analysis of whole cell lysates.</p