Analysis of the Transforming growth factor-β1 signaling network.

<p><b>A,</b> Expression of TGF-β1 mRNA and <b>B,</b> secreted TGF-β1 protein for assessment of the autocrine TGF-β feedback loop. Bars represent the mean+SEM of 6 independent experiments, 3 replicates each. <b>C,</b> Expression of CTGF mRNA. Quantitative real-time PCR analysis of mRNA transcripts. Bars represent the mean+SEM of 6 independent experiments quantified with the ΔΔCt method in duplicates. <b>D,</b> Expression of TGF-β receptor 1 protein. <b>E-G,</b> Induction of canonic and non-canonic TGF-β signaling. <b>E,</b> Phosphorylation of SMAD3 at Ser423/425. <b>F,</b> Phosphorylation of AKT at Ser473. <b>G,</b> Phosphorylation of ERK1/2 at Thr202/Tyr204. Analysis of all experiments was performed after 6 days treatment with 10 ng/ml transformin growth factor -β1. Representative western blots of 6 independent experiments are shown. Bars represent the mean+SEM of densitometrically determined band intensities after normalization to GAPDH (TGFBR1, pSMAD3) or total kinase (pAKT, pERK). Control was set to 1. *P<0.05, **P<0.01, ***P<0.001.</p

Measurement of functional L-type Cav1.2 Ca2+ channels in response to growth factors.

<p>Functional L-type Cav1.2 Ca²⁺ channels measured as nimodipine sensitive Ca²⁺-influx upon depolarisation with 60 mmol/L KCl in response to treatment with systemic sclerosis microenvironment defining growth factors for 6 days. <b>A,</b> Bars represent the mean+SEM of nimodipine sensitive Ca²⁺-influx from 3 independent experiments. Control was set to 1. *P<0.05, **P<0.01, ***P<0.001. <b>B-F,</b> Representative recordings of Ca²⁺ dependent intracellular fluorescence intensities in response to depolarisation with KCl. Mean±SEM F/F0 of 30–40 cells per growth factor are plotted against time. Black lines represent tracings without the specific L-type calcium channel blocker nimodipine, grey lines those obtained after preincubation with 1 mmol/L nimodipine.</p

Cell functions of mesenchymal stromal cells upon treatment with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Chemotactic response towards a 5 ng/ml gradient of each growth factor. Bars represent the mean+SEM of 3 independent experiments, 3 replicates each. Control was set to 1. <b>B,</b> Proliferation measured by BrdU incorporation after stimulation with 10 ng/ml of each growth factor for 24 h. Bars represent the mean+SEM of 3 independent experiments, 5 replicates each. Control was set to 1. <b>C,</b> Total collagen content in cell culture supernatants after 6 days of treatment with 10 ng/ml of each growth factor. Bars represent the mean+SEM of 6 independent experiments, 2 replicates each. *P<0.05, **P<0.01, ***P<0.001. <b>D,</b> Illustration of the induction of specific cell types of vascular smooth muscle cell (VSMC) and fibroblast lineages by systemic sclerosis microenvironment defining growth factors.</p

Expression of contractile proteins in response to treatment with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> SM22α <b>B,</b> smooth-muscle calponin (sm-Calponin) <b>C,</b> myosin-light-chain-kinase (MLCK) <b>D,</b> smooth muscle-α-actin (sm-Actin). Bars represent the mean+SEM of densitometrically determined band intensities after normalization to α-tubulin. Densitometric quantification and representative western blots of 6 independent experiments are shown. Cells hat been treated for 6 days. Control was set to 1. *P<0.05, **P<0.01, ***P<0.001.</p

Phenotypic modulation upon long-term stimulation (6 days) with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Representative phase contrast photomicrographs from a total of 6 independent experiments are shown. (original magnification x200, scale bar = 50 μm) <b>B,</b> Representative immunofluorescence images from a total of 6 independent experiments are shown. (green = smooth muscle-α-actin, blue = nuclear DNA stained with 4',6-diamidino-2-phenylindole (DAPI), original magnification x400, scale bar = 50 μm).</p

Characterization of mesenchymal stromal cells from healthy controls and patients with systemic sclerosis.

<p><b>A,</b> Representative FACS analysis of an MSC defining surface marker panel confirming homogeneity of isolated cells. <b>B,</b> Osteoblastic differentiation of MSCs after incubation for 21 days with osteoblast-induction medium. (Alizarin red S staining, original magnification x100, scale bars = 50 μm) <b>C,</b> Adipocytic differentiation of MSCs after incubation for 21 days with adipocyte-induction medium. (Oil-Red-O staining, original magnification x200, scale bars = 50 μm) <b>D,</b> Chondroblastic differentiation of MSCs after incubation for 32 days with chondroblast induction medium. Western blot analysis for chondrocyte specific type II collagen.</p