Description of SSc patients from whom skin biopsies were obtained.

<p>Description of SSc patients from whom skin biopsies were obtained.</p

DOK5 is upregulated in lung and skin tissues engineered to express IGFBP-5 and localizes to cell nuclei <i>in vivo</i>.

<p><b>A.</b> DOK5 expression was examined in skin (left panel) and lung (right panel) tissues of C57BL6/J mice treated with control or IGFBP-5 expressing adenovirus (control AdV and IGFBP-5 AdV, respectively). DOK5-expressing cells appear brown following AEC staining. Magnification, 800x. Data are representative of skin and lung tissues from 2 mice/group. <b>B.</b> Graphical presentation of data shown in A using 8 fields per treatment condition. Y axis shows the number of DOK5-positive cells counted at a magnification of 400X. Bars denote the mean and error bars denote SD.</p

DOK5 expression increases dermal thickness.

<p>A. Representative results of organ culture of human skin engineered to express IGFBP-5, DOK5, or control. DOK5 expression was examined in human skin treated with control or IGFBP-5 expressing adenovirus (control AdV and IGFBP-5 AdV, respectively). DOK5-expressing cells were shown in red using AEC staining. Magnification, 800x. Results are representative of three experiments. B. DOK5 triggers dermal fibrosis. Dermal thickness and collagen content as assessed by Masson Trichrome are increased in DOK5-treated human skin. Magnification, 40x. Findings were replicated using skin from two different donors. C. DOK5 induces a significant increase in dermal thickness. Dermal thickness was measured in human skin and represents the average of 4 experiments. Values shown represent mean and SD. *<i>P</i><0.008.</p

Description of SSc patients from whom lung tissues were obtained.

<p>Description of SSc patients from whom lung tissues were obtained.</p

DOK5 expression is upregulated by endogenous and exogenous IGFBP-5 via MAPK.

<p>A. DOK5 mRNA is upregulated by endogenous IGFBP-5. Human skin fibroblasts were infected with adenovirus expressing IGFBP-3, IGFBP-5 or control (IGFBP-3 AdV, IGFBP-5 AdV, or control AdV, respectively) at an MOI of 50. Expression of DOK5 was examined by RT-PCR. β-actin is shown as a loading control. Results are representative of two independent experiments using different fibroblast strains. B. Production of DOK5 protein is induced by endogenous IGFBP-5. Production of DOK5 was examined by immunoblotting in whole cell lysates treated as in A. Molecular weight of DOK5 is ∼30 kDa. GAPDH is shown as a loading control. The experiment was repeated twice with reproducible results. C. Production of DOK5 protein is induced by exogenous IGFBP-5. Human skin fibroblasts were stimulated with recombinant IGFBP-5 (rBP5), and whole cell lysates were harvested. DOK5 production was examined by immunoblotting. GAPDH is shown as a loading control. The experiment was repeated three times. D. Graphical presentation of the data shown in panel C. *indicates p<0.05. E. Induction of DOK5 protein production is blocked by the Mek inhibitor (MEKi). Meki was administered after infection with AdVs and levels of DOK5 examined by immunoblotting. GAPDH is shown as a loading control. Data shown are representative of two independent experiments.</p

DOK5 mRNA and protein levels are increased in lung and skin tissues of SSc patients.

<p>A. DOK5 protein is increased in lung tissues of SSc patients. DOK5 protein was detected in total lung tissue homogenates of patients with SSc and healthy controls using immunoblotting. B. DOK5 protein levels are significantly elevated in SSc lung tissues. Data presented show average signal intensity of DOK5 in lung homogenates from 3 patients with SSc and 3 healthy controls. Protein levels were examined using immunoblotting and signals quantified with densitometry. *<i>P</i><0.05. C. Representative images of DOK5 in skin tissue from a patient with SSc and a normal control (NC). DOK5 was detected using immunohistochemistry. Brown signal indicates DOK5. Arrows point to representative cells positive for DOK5. Sections were counter-stained with hematoxylin. Images are representative of sections from three patients and three controls. Bar = 20 µm. Magnification, 400x. D. Localization of DOK5 and αSMA in SSc skin. White color indicates merging of blue, red and green, and shows expression of DOK5 in nuclei. Images are representative of immunofluorescence staining done on skin samples from two patients with SSc and two controls. Bar = 20 µm.</p

DOK5 localizes to the nucleus.

<p>A. Confirmation of DOK5 expression. Expression of DOK5 was confirmed by immunoblotting following transient transfection of COS7 cells with DOK5-pAdlox or control-pAdlox plasmids. GAPDH is shown as a loading control. Data were confirmed in an additional two experiments. B. DOK5 translocates to the nucleus. DOK5 localization in COS7 cells treated as in A using immunofluorescence. DOK5 is shown in red and nuclei stained with DAPI appear blue. Merged signal appears purple. Indicated times reflect hours post-transfection. Magnification, 800x. C. Nuclear DOK5 levels are increased by endogenous IGFBP-5. Lysates of primary human fibroblasts infected with adenovirus (AdV) were fractionated into cytoplasmic and nuclear fractions and induction of DOK5 was examined using immunoblotting. β-actin and Histone H3 are shown as loading control for cytoplasmic and nuclear fractions, respectively. Findings were confirmed in two independent experiments. D. Nuclear DOK5 levels are induced by exogenous IGFBP-5. Cytoplasmic fractions of primary human fibroblasts treated with recombinant IGFBP-5 (rBP5) were harvested and induction of DOK5 was examined. Histone H3 was used as a loading control. Findings were confirmed in two independent experiments.</p

Additional file 3: Figure S2. of Identification of definitive serum biomarkers associated with disease activity in primary Sjögren’s syndrome

Serum levels of five proteins in pSS, sSS, sicca syndrome and HCs. The five proteins were CXCL13, TNF-R2, CD48, BAFF and PD-L2. Primary SS (pSS), n = 58; secondary SS (sSS), n = 6; other sicca syndrome, n = 13; healthy controls (HCs), n = 38. Differences in quantitative variables were analyzed by the Mann-Whitney U test when comparing two groups and by the Kruskal-Wallis test when comparing multiple groups. *P value <0.05, which was considered significant. (TIF 33972 kb

Additional file 2: Figure S1. of Identification of definitive serum biomarkers associated with disease activity in primary Sjögren’s syndrome

Functional annotation of differentially expressed proteins in pSS patient sera. Nodes indicate molecular concepts or set of biologically related genes. Name of each node is indicated in black text on the node. The node size represents the proportion of differentially expressed gene symbols in the concepts (e.g., the “chemokine signaling pathway” and “extracellular region” concepts contain 14 and 58 genes, respectively). Length of lines between nodes represents degree of overlap between symbols. Colored lines indicate strength of functional relationship from strong to weak, as follows: red, yellow, green and gray. Green nodes indicate immune response-related molecular concepts, and red nodes indicate platelet-related molecular concepts. (TIF 9752 kb

Additional file 1: Figure S1. of Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease

Flow cytometric analysis of the percentage of circulating follicular helper T (Tfh) cells and activated Tfh cells. The percentage of Tfh cells (A) and activated Tfh cells (B) from patients with IgG4-related disease (IgG4-RD) (n = 17), primary Sjögren’s syndrome (pSS) (n = 20), multicentric Castleman’s disease (MCD) (n = 5), and healthy controls (HC) (n = 12). *P < 0.05; **P < 0.0001 for analysis using the Kruskal-Wallis test, followed by group-wise comparisons using the Mann-Whitney U test. (TIF 42 kb