Mechanism inducing endodermal cell differentiation.

<p>(A) RT-PCR of different lineage-specific marker genes in wt and <i>G6pdΔ</i> ES cells in presence of a lower oxygen concentration (5%) and in normal culture conditions (20%). (B) RT-PCR of different lineage-specific markers in differentiated wt E14 and <i>Pgd</i>+/− ES cells at 8, 10 and 13 days of neural differentiation. (C) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in <i>Pgd</i>+/− ES cells. Scale bars, 75 µm. (D) qRT-PCR for Sox17 and GATA4 in wt and <i>G6pdΔ</i> ES cells at day 10 after treatment with D-(-)-ribose during neural differentiation. Values are means ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Endodermal induction in <i>G6pdΔ</i> ES cells.

<p>(A) Analysis of different markers in wt and <i>G6pdΔ</i> ES cells during neural differentiation. Expression profiles of undifferentiated ES cells (Oct4 and Nanog), neural precursors (Nestin), neurons (NF-L), astrocytes (GFAP), mesendodermal precursors (GATA4), endodermal precursors (Sox17), and cardiac precursors (Nkx2.5) markers were analyzed by RT-PCR. RNA was isolated from cells at different days of differentiation. Lane C, positive control, RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (B) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in <i>G6pdΔ</i> ES cells. Scale bars, 50 µm. (C) RT-PCR analysis of GATA4, Sox17, NF-L (neural marker), TH (dopaminergic neuron marker) and GAD65 (gabaergic neuron marker) on wt, two different <i>G6pdΔ</i> ES cell lines, and <i>G6pdΔ</i>^pG6pd during differentiation. Lane C, positive control, on RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (D) Western blot analysis with anti-Cripto and anti-Actin antibodies performed on protein extracts from wt and <i>G6pdΔ</i> ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated. (E) Western blot analysis with anti-phospho-Smad2 and anti-Actin antibodies performed on protein extracts from wt and <i>G6pdΔ</i> ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated.</p

Definitive and extraembryonic endoderm differentiated from <i>G6pdΔ</i> ES cells.

<p>(A) Immunofluorescent staining of the differentiated mouse <i>G6pdΔ</i> ES cells for Sox17 (red) and nuclei (blue) at 8 days of neural differentiation. PH, phase contrast images. Scale bars, 25 µm. (B) RT-PCR analysis of the endodermal markers GATA4 and extraembyonic endodermal markers Sox7 during differentiation. Lane C, positive control, RNA isolated from embryos and yolk sacs at 9,5 dpc. Amplified HPRT is shown as a positive control. (C) qRT-PCR for Pdx1 in wt and <i>G6pdΔ</i> ES cells at 13 days after treatment with Indolactam V from day 8 during differentiation. Values are means ± SD (n = 2). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

VCAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: Time course of induction of VCAM1 transcripts, in EAhy926 endothelial cells, by treatment with 1 µmol/L homocysteinylated albumin. Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin. (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin. (p<0.001).</p

Amplification conditions and primer pairs for PCR experiments.

<p>All conditions are relevant to real time PCR except for <i>VCAM1</i>, where tradition PCR has been employed. 35 amplification cycles have been performed.</p

ICAM1 expression in endothelial cells treated with N-homocysteinylated albumin.

<p>Panel A: expression levels of ICAM1 transcripts quantitated by real time PCR (treated: 1 µmol/L homocysteinylated albumin; control: unmodified albumin); (p<0.001). Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin; (p<0.001).</p

Characterization of homocysteinylated albumin by mass spectrometry.

<p>Panel A: ESI-MS of human serum albumin. Panel B: ESI-MS of homocysteinylated albumin. Inset: magnification on expanded scale of the signal at Da = 67805. The family of molecular ions is compatible with the structures shown in the panel.</p

Time-dependent, increased expression of ADAM17, MCP1 and Hsp60 in endothelial cells upon homocysteinylated albumin treatment.

<p>Panel A: Real time PCR evaluation during time course of <i>ADAM17</i>, <i>MCP1</i> and <i>Hsp60</i> mRNA. Panel B: ELISA assay of MCP1 released in the culture medium of treated cells. Panel C: Western blotting analysis of intracellular levels of ADAM17, and Hsp60, and analysis of Hsp60 released in the medium by immunoprecipitation and western blotting (Hsp60 IP). A: unmodified albumin control; AH: homocysteinylated albumin treatment. Levels of transcripts or proteins in the homocysteinylated albumin sample group were significantly increased compared to control (p<0.001).</p

Validation by Q-PCR of transcriptome results relevant to upregulated gene involved in endothelial dysfunction.

<p>A: unmodified albumin; AH: homocysteinylated albumin. Gene expression in the AH sample group was significantly increased with respect to the corresponding genes in the A sample group (p<0.001).</p

Effects of homocysteinylated albumin on monocyte adhesion.

<p>U937 monocytoid cells adhesion onto an endothelial monolayer (EAhy926) expressed as adherent cells (number/field; panel A) and percentage adherent cells compared to positive control (panel B). Counts are the mean of ten independent experiments, each carried out by counting five different fields/sample of triplicate samples. Examples of microscopic fields are shown on the right. C: negative control (untreated cells); A: unmodified albumin; AH: homocysteinylated albumin; AC: carboxymethylated albumin; T: positive control (Tnf-α). 0.3 or 1: homocysteine micromolar concentration present in the assay in the form of <i>N</i>-homocysteinyl groups bound to albumin, as comparable to the in vivo situation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031388#pone.0031388-Perna1" target="_blank">[14]</a>; p<0.0001.</p