NOS inhibition by L-NAME suppresses cell protection by SR 11302 during cholestasis.

<p>Effect of inhibition of NOS activity on (A) cell doubling time (n = 6) and cell metabolic activity (n = 8); (B) caspase-3-associated activity (n = 3); and (C) cyclin D1 expression (n = 4). SR 11302 was used at 50 μM. In panel (A), only positive data for cell doubling time were used (n = 4 for group ‘GCDCA + SR 11302’, and n = 3 for group ‘GCDCA + SR 11302 + L-NAME’). Data expressed as mean ± SE. Statistically significant difference versus control group* or versus GCDCA group^# or between groups ‘GCDCA + SR 11302’ and ‘GCDCA + SR 11302 + L-NAME’ (S) are marked.</p

NOS-3 activity recovery by AP-1 inhibition is associated with cell survival during experimental cholestasis.

<p>Effects of AP-1 inhibition on (A) accumulation of NO-end products (n = 3), (B) caspase-3-associated activity (n = 4), (C) cell proliferation and (D) dead cell count (n = 3). In panel (B), SR 11302 was assayed at 50 and 10 μM. When no indicated, SR 11302 was used at 50 μM. Data expressed as mean ± SE. Statistically significant difference versus control group* or versus GCDCA group^# are marked.</p

AP-1 downregulates NOS-3 expression during cytotoxicity by GCDCA.

<p>Cells not exposed (control group) / exposed to GCDCA (GCDCA group) were treated with Curcumin, Quercetin or the synthetic retinoid SR 11302. (A) Representative western blot for phospho-SP1 (Thr453) (Curcumin and Quercetin, n = 3; SR 11302, n = 7). (B) NOS-3 promoter activity (Curcumin and Quercetin, n = 6; SR 11302, n = 3). (C) NOS-3 protein expression (n = 3). (D) Dose-dependent NOS-3 expression recovery by SR 11302 during GCDCA cytotoxicity. SR 11302 was assayed at 50, 10 and 1 μM. When no indicated, SR 11302 was used at 50 μM. Densitometry analysis for panels (A), (C) and (D) are shown. Data expressed as mean ± SE versus control group. Statistically significant difference versus Control group* or versus GCDCA group^# are marked.</p

Expression of transcription factors cJun and cFos in GCDCA-treated HepG2.

<p>Effect of the administration of MnTBAP or MitoQ. (A) Representative western blots for cJun (n = 3), phospho-cJun (Ser63) (n = 4), cFos (n = 5) and phospho-cFos (Ser374) (n = 4). Β-Actin was used as loading control. (B) Densitometry analysis of blots included in panel A. In the densitometry analysis, the + sign indicates GCDCA administration plus the showed treatment. Data as mean ± SE versus control group. Statistically significant difference versus control group* or versus GCDCA group^# are marked.</p

Confirmation of DIGE results by western-blot.

<p>Detection and quantification of (A) carboxypeptidase-N (CPN), (B) serum paraoxonase /arylesterase 1 (PON1), and (C) ceruloplasmin (CP) in depleted human plasma samples previously analysed by DIGE. Protein band density was calculated by <i>QuantityOne 4</i>.<i>4</i>.<i>0</i> (BioRad) software. Results are shown as arbitrary units (AU); (D) Transferred proteins to the nitrocellulose membrane were detected by Ponceau S stain as western-blot loading control. C1, C2 and C3: protein samples of patients from the control group; T1, T2 and T3: protein samples of patients from the tumor group.</p

Multivariate logistic regression analysis for the candidate biomarkers.

<p>Complement component 4a was the only independent predictor of HCC after controlling for possible confounding factors.</p><p>*OR and confidence interval for C4a has been calculated for an increment of 0.5 ng/mg plasma protein.</p><p>C4a: Complement component 4a; CPN: carboxypeptidase-N; PON1: serum paraoxonase /arylesterase 1; CP: ceruloplasmin; FGA: fibrinogen-alpha; IgM: immunoglobulin mu chain C region.</p><p>Multivariate logistic regression analysis for the candidate biomarkers.</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

Primers used for quantitative RT-PCR analyses.

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