Genes differentially expressed in SSc tenosynovial ...

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Genes differentially expressed in SSc tenosynovial ... Genes differentially expressed in SSc tenosynovial compared with synovial tissue samples from patients with inflammatory joint disorders. Student’s t-tests indicated that 2549 transcripts were significantly overexpressed in SSc compared with OA samples, with a fold-change >2. Pathway analyses showed that these transcripts were significantly enriched in the indicated GOTERM pathways (all pathways and enrichment scores are listed in supplementary Table S1, available at Rheumatology online). The data are displayed as log2-transformed, mean-centered values. MIC: microcrystalline arthritis; PSO: psoriatic arthritis; USP: ubiquitin-specific peptidase. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Rheumatology (Oxford), Volume 58, Issue 4, 03 January 2019, Pages 708–718, https://doi.org/10.1093/rheumatology/key401 The content of this slide may be subject to copyright: please see the slide notes for details.

Biopsies were obtained from clinically ... Biopsies were obtained from clinically affected and unaffected skin regions in 20 patients with diffuse SSc and 10 healthy controls. (A) Characteristic images of USP15, USP16, USP37 and USP48 stains, showing positive fibroblasts, endothelial and some inflammatory cells, mainly in perivascular areas. (B) Immunoquantification show significant overexpression of the stained USPs in SSc compared with healthy skin samples. *P < 0.05; **P < 0.005; ***P < 0.0005 using Mann–Whiney tests. USP: ubiquitin-specific peptidase. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Rheumatology (Oxford), Volume 58, Issue 4, 03 January 2019, Pages 708–718, https://doi.org/10.1093/rheumatology/key401 The content of this slide may be subject to copyright: please see the slide notes for details.

Synovial fibroblasts (n = 6 cell ... Synovial fibroblasts (n = 6 cell lines), obtained from the knee of patients with OA, were cultured for 24 h in duplicate wells with DMOG, a hypoxia-inducible factor 1α hydroylase inhibitor, or TGF-β. Expression of the indicated USP was evaluated by quantitative PCR. Results are displayed as mean (± s.e.m.) fold-changes compared with unstimulated cells from three different experiments. *P < 0.01; **P < 0.005; ***P < 0.0001 using unpaired Student’s t-tests. Mean value for USP37 induction by DMOG, 19.1. DMOG: dimethyloxalylglycine; ND: not done; USP: ubiquitin-specific peptidase. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Rheumatology (Oxford), Volume 58, Issue 4, 03 January 2019, Pages 708–718, https://doi.org/10.1093/rheumatology/key401 The content of this slide may be subject to copyright: please see the slide notes for details.

Fig. 4 Effects of USP inhibition on SMAD3 phosphorylation and collagen gene expression in TGF-β-stimulated ... (A, B) Total and phospho-SMAD3 western blots in unstimulated or TGF-β-stimulated synovial fibroblasts, cultured in multiplicate wells in the presence or the absence of NSC632839, a pan-USP inhibitor. (A) Representative experiment. (B) β-actin-normalized SMAD3 and phospho-SMAD3 quantification in four different experiments. P-values were calculated using paired Student’s t-tests. *P < 0.01; ***P < 0.0001. (C) Quantitative PCR evaluation of COL1A1, COL3A1 and fibronectin (FN1) gene expression in unstimulated or TGF-β-stimulated synovial fibroblasts, cultured in duplicate wells in the presence or the absence of NSC632839. Results are displayed as mean (± s.e.m.) fold-changes in β2-microglobulin-normalized gene expression compared with unstimulated cells, from four different experiments. P-values were calculated using paired Student’s t-tests. **P < 0.005; ***P < 0.0001. USP: ubiquitin-specific peptidase. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Rheumatology (Oxford), Volume 58, Issue 4, 03 January 2019, Pages 708–718, https://doi.org/10.1093/rheumatology/key401 The content of this slide may be subject to copyright: please see the slide notes for details.

(A, B) Total SMAD3 was ... (A, B) Total SMAD3 was immunoprecipitated from unstimulated or TGF-β-stimulated synovial fibroblasts, cultured in duplicate wells in the presence or the absence of NSC632839. All cells were pre-incubated with epoxomicin, in order to inhibit proteosomal degradation of ubiquitinated SMAD3. The presence of ubiquitinated chains was studied by western blot (Ub-SMAD3) and normalized for total SMAD3 in the immunoprecipitates. (A) Representative experiment. (B) Total SMAD3-normalized quantification of SMAD3 ubiquitination. The results are displayed as the mean (± s.e.m.) from three different experiments. *P < 0.05 using unpaired Student’s t-tests. (C, D) Total and phospho-SMAD3 western blots (C) and β-actin-normalized quantification (D) in unstimulated or TGF-β-stimulated synovial fibroblasts, pre-incubated with epoxomicin, and cultured in duplicate wells in the presence or the absence of NSC632839. Results are displayed as the mean (± s.e.m.) values from three different experiments. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Rheumatology (Oxford), Volume 58, Issue 4, 03 January 2019, Pages 708–718, https://doi.org/10.1093/rheumatology/key401 The content of this slide may be subject to copyright: please see the slide notes for details.

(A, B) Human ... (A, B) Human synovial fibroblasts were left non-transfected, or transfected with control or USP15 siRNA, and then stimulated with TGF-β. (A) Representative USP15, phospho-SMAD3 and total SMAD3 western blot. (B) β-actin-normalized USP15, total SMAD3 and phospho-SMAD3 quantification in two different experiments. P-values were calculated using unpaired Student’s t-tests. *P < 0.01, ***P < 0.0001. (C, D) Human embryonic kidney (HEK293) cells were transfected with a control or USP15-encoding plasmid, and stimulated in duplicate wells with TGF-β. (C) Representative USP15, phospho-SMAD3 and total SMAD3 western blot. (D) β-actin-normalized USP15, total SMAD3 and phospho-SMAD3 quantification in three different experiments. P-values were calculated using unpaired Student’s t-tests. **P < 0.005. USP: ubiquitin-specific peptidase. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Rheumatology (Oxford), Volume 58, Issue 4, 03 January 2019, Pages 708–718, https://doi.org/10.1093/rheumatology/key401 The content of this slide may be subject to copyright: please see the slide notes for details.