Primers used for quantitative RT-PCR analyses.

<p>Primers used for quantitative RT-PCR analyses.</p

Differential stem cells markers in undifferentiated and differentiated human mesenchymal stem cells.

<p>Levels of CD13, CD49e, CD166, CD133 and VEGFR2 in undifferentiated cells (UC), CM1 and CM2-treated cells after 21 days of culture. (Conditioned medium: CM). Values are expressed as mean of percentage ± standard deviation. (a p<0.001 and b p<0.01 vs. CM1-treated cells; +++ p<0,001 and + p<0,05 vs. undifferentiated cells).</p

The treatment of human mesenchymal stem cells with CM2 induces nuclear translocation ofβ-catenin and Wnt signaling activation.

<p>A) To determine β-catenin subcellular localization, human mesenchymal stem cells undifferentiated (UC) and treated with conditioned medium 1 (CM1) or 2 (CM2) after 21 days of culture were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue). Merged image of β-catenin-FITC and DAPI staining is also shown. Original magnification: 40×. B) mRNA expression of Lrp5/6, Frizzled- 3 (FZD3) and c-myc was evaluated in undifferentiated cells and cells treated with conditioned medium 1 (CM1) or 2 (CM2). Fold of undifferentiated cells at 21 days of culture. ^a p<0.001 vs. CM1-treated cells. C) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034656#pone-0034656-g004" target="_blank">Figure 4</a> c shows western blot of p53 and α-tubulin as loading control. Image is representative of three independent experiments.</p

Comparative analysis by DIGE of proteins differentially expressed in hepatocytes obtained with CM1 or CM2 differentiation protocols.

<p>Cyt: Cytoplasm; Nuc: Nucleus; Secr: Secreted protein; ER: Endoplasmic reticulum; Mit: Mitochondria; Lys: Lysosome.</p

Stem cells/cancer stem cells markers expression in undifferentiated cells at time 0 days.

<p>Data are included as mean ± standard deviation.</p

The activation of Wnt/β-catenin during hepatocyte differentiation is associated with the presence of related proteins to tumoral phenotype.

<p>Relative abundance of specific proteins (DIGE analysis) in human mesenchymal stem cells undifferentiated after 21 days of culture (UC21d) and in mesenchymal stem cells differentiated into hepatocytes with conditioned medium 1 (CM1) or 2 (CM2). B) Western blot confirmation of the changes observed by DIGE analysis in the abundance of some proteins in CM1 and CM2 hepatocytes: Adenine phosphoriobosyl transferase (APT), cathepsin B precursor (CATB), L-lactate dehydrogenase β chain (LDHB), transgelin (TGL2), tropomyosin β chain (TPM2) and nuclear β-catenin. Tubulin and TFIIB were used as cytoplasm and nuclear loading control respectively.</p

The treatments with CM1 or CM2 increase the expression of hepatospecific genes in human mesenchymal stem cells.

<p>Relative levels of mRNA expression of A) albumin (ALB), B) α-fetoprotein (αFP), C) α1-antitrypsin (α-1-AT), D) CCAAT/enhancer-binding protein beta (C/EBP) and E) cytochrome P450 (CYP3A5) were determined in human undifferentiated mesenchymal stem cells before and after differentiation with conditioned medium 1 (CM1) or 2 (CM2) after 7, 14 and 21 days of culture; Gene expression is shown as fold-changes compared to undifferentiated cells at each time. Values are expressed as mean ± standard deviation. All genes were increased significantly respect to undifferentiated cells (UC). ^a p<0.001, ^b p<0.01 vs. CM1 or CM2.</p

Reduced liver fibrosis in SPARC deficient mice.

<p>(A) Representative photomicrographs of liver sections stained with picrosirius red. SPARC^+/+ mice show staining limited to periportal areas (left panel), while liver sections from TAA-treated SPARC^+/+ mice exhibits marked portal fibrosis and portal-portal bridges (central panel) and those from TAA-treated SPARC^−/− mice present weak fibrotic response (right panel). (B) Morphometric quantification of Sirius red stained area showing a significant attenuation of the fibrotic process in TAA-treated SPARC^−/− mice when compared to treated wild-type mice. **p<0.01, Mann-Whitney test. (C–D) Quantitative data from qPCR analysis of collagen (COL1A2) and MMP-2 mRNA expression. *p<0.05, **p<0.01 versus SPARC^+/+ TAA 10 weeks, Mann-Whitney test. (E) Representative pictures taken from liver sections of from SPARC^+/+ or SPARC^−/− mice, at 7 days after BDL. Original magnification 200X. PT, portal tract; CV, central vein. (F) Morphometric quantification of Sirius red stained area showing a significant attenuation of the fibrotic process in SPARC^−/− mice at day 7 after BDL when compared to wild-type mice. **p<0.01, Mann-Whitney test. (G) qPCR analysis of collagen mRNA expression in SPARC^+/+ and SPARC^−/− mice subjected to BDL. *p<0.05, versus BDL SPARC^+/+ Mann-Whitney test.</p

Induction of SPARC mRNA expression during liver fibrogenesis.

<p>(A) Quantitative data showing differences in SPARC mRNA expression levels in human cirrhotic (Pt#1 to Pt#5) with fibrosis degree F4 and non-cirrhotic liver samples as measured by qPCR. *p<0.05, **p<0.01 compared with healthy liver samples. Mann-Whitney test. (B) qPCR analyses of liver samples from TAA and BDL mice. Complementary DNA was synthesized and was subjected to qPCR for the expression of SPARC transcripts. The relative amount of the PCR product (AU, arbitrary units) amplified from control liver samples was set at 1. *p<0.05 versus control (−TAA), **p<0.01 versus control (−BDL), Mann-Whitney test.</p

IPA® top molecules which were significantly alters in SPARC<i>^−/−</i> versus SPARC^+/+ mice and those modified in SPARC<i>^−/−</i> versus SPARC^+/+10 weeks TAA treated mice.

<p>IPA® top molecules which were significantly alters in SPARC<i>^−/−</i> versus SPARC^+/+ mice and those modified in SPARC<i>^−/−</i> versus SPARC^+/+10 weeks TAA treated mice.</p