Patient characteristics.

<p>Abbreviations: f:female, m:male, ANA: anti-nuclear antibodies; ACA: anti-centromere antibodies; nd: no data, AZA, azathioprine, Cyc: cyclophosphamide, MMF: mycophenolate mofetil, MTX: methotrexate.</p

Healthy monocytes can differentiate into α-SMA and collagen expressing cells upon GM-CSF stimulation.

<p>A) CD14⁺ monocytes from healthy donors were cultured for 14 days with GM-CSF, IL-4 or ET-1. Western blot analysis showed expression of type-1 collagen and α-SMA under distinct conditions. Fibroblasts were used as a positive control, GAPDH was used as loading control. B) Monocytes were treated with GM-CSF and 0–500 ng/ml of ET-1 over 14 days. Western Blot analysis for type-1 collagen and α-SMA was conducted. GAPDH was used as loading control. C) and D) Quantification of the western blots from 3 independent experiments are shown. E) Expression of CD14, HLA-DR and CD11c was assessed by flow cytometry.</p

Patient monocytes need less stringent stimulation to upregulate α-SMA.

<p>A) Monocytes from healthy controls or SSc patients were cultured for 14 days with GM-CSF, IL-4 or ET-1. α-SMA levels were detected by Western blot analysis. GAPDH was used as loading control. B) Quantification of Western blots from 8 patients and 8 healthy controls showed stronger expression of α-SMA in SSc monocytes compared to healthy controls despite no significant difference could be detected. C) qPCR analysis of the procollagen expression in GM-CSF treated monocytes from healthy controls (HC) (n = 4) or SSc patients (n = 8). Data were normalized to expression of 18S. (p = 0.481). D) and E) Monocytes from HC (n = 4) and SSc patients (n = 3) were treated with various combinations of GM-CSF, IL-4 and ET-1. Procollagen expression was measured by qPCR. Data were normalized to expression of 18S. The concentration of ET-1 in all experiments was 100 ng/ml. * Statistically significant (P-value<0.05).</p

Morphological analysis of monocytes from SSc patients and healthy controls.

<p>A) Immunofluorescence staining of α-SMA (red) in GM-CSF treated monocytes from healthy control or a SSc patient. Fibroblasts were used as positive control. Nuclei were stained with DAPI (blue). For quantification, spindle shaped cells per 5×5 cm field were counted and normalized to cell numbers (B). ** Statistically highly significant (P-value<0.01).</p

Levels of col1A1 and TIMP-1 mRNA and protein expression after miR-29a transfection.

<p>HC dermal fibroblasts were transfected with 30 nM miR-29a, 30 nM non-targeting cont-miR or vehicle only (untr). Col1A1 (A) and TIMP-1 mRNA (B) and TIMP-1 protein secretion (C) were determined after 6 h and 24 h following transfection. TIMP-1 protein level was also determined after 24 h from transfection and following 1 µg/ml TLR4 agonists (LPS) treatment (D). Densitometry analysis of three independent experiments of HC fibroblasts after 24 h from transfection was performed and GAPDH was used as a loading control (D).</p

Functional effect of miR-29a on contractile properties of HC fibroblasts.

<p>HC dermal fibroblasts were transfected with 30 nM miR-29a, 30 nM cont-miR or stimulated with 100 ng/ml exogenous recombinant TIMP-1 and contractile properties of HC dermal fibroblasts were assessed after 24 h by collagen degradation assay (A) and percentage of collagen gel area was measured (B). MMP-1 mRNA was measured in untreated HC dermal fibroblasts cultivated on monolayers or seeded on collagen gel (C). Secreted MMP-1 level was measured in HC dermal fibroblasts cultivated on monolayers or seeded on collagen gel in the presence of miR-29a, cont-miR or recombinant TIMP-1 (D). Similarly, TIMP-1 secretion was measured in HC dermal fibroblasts seeded on collagen gel in the presence of miR-29a, cont-miR or vehicle only (untr) (E).</p

Sequence of 3′UTR of TAB1 to which Target protector is complementary (seed regions of 3′UTR of TAB1 are highlighted in bold).

<p>Sequence of 3′UTR of TAB1 to which Target protector is complementary (seed regions of 3′UTR of TAB1 are highlighted in bold).</p

TAK1/TAB1 inhibitor downregulates TIMP-1 secretion in TGF-β stimulated HC dermal fibroblasts.

<p>HC dermal fibroblast were pre-treated for 2 h with graded doses of Oxozeanol (TAK1/TAB1 inhibitor) prior to TGF-β stimulation (5 ng/ml) and TIMP-1 secretion and mRNA level were measured (A, B). To assess viability of HC dermal fibroblasts upon 2 µM Oxozeanol treatment, MTS assay was performed (C). HC dermal fibroblasts were transfected with 100 nM siRNA against TAB1 or scrambled oligos and TAB1 protein and TIMP-1 secretion were analysed after 48 h from knockdown (D, E).</p

Target protectors induce profibrotic phenotype in HC dermal fibroblasts.

<p>HC dermal fibroblasts were transfected with 30 nM miR-29a, 30 nM cont-miR and TAB1 mRNA (A) and protein (B) expression were measured after 24 h. In addition, HC dermal fibroblasts were stimulated with 5 ng/ml TGF-β as a positive control for TIMP-1 induction. Densitometry analysis of four independent experiments was performed and GAPDH was used as a loading control (B). HC dermal fibroblasts were transfected with 500 nM TP A, TP B, or both TP A+B and TIMP-1 secretion was measured after 24 h (C). The basal level of TAB1 mRNA in HC and SSc fibroblasts (D).</p

miR-29a reverses phenotype of SSc dermal fibroblasts.

<p>The basal level of col1A1 was compared between HC and SSc fibroblasts (A). SSc dermal fibroblasts were transfected with 30 nM miR-29a, 30 nM cont-miR or stimulated with 5 ng/ml TGF-β and col1A1 mRNA level was measured (B). HC and SSc dermal fibroblasts were transfected with 30 nM miR-29a, 30 nM cont-miR or stimulated with 5 ng/ml TGF-β and secreted TIMP-1 was measured (C). Schematic representation of the role of miR-29a in ECM regulation via TAB1 and TIMP-1 suppression in HC and SSc dermal fibroblasts (D). Enhanced activation of TGF-β signaling in SSc dermal fibroblasts compared to HC fibroblasts results in downregulation of miR-29a, which leads to increased expression of miR-29a target gene - TAB1.</p