Cell viability after MGO treatment.

<p>PC12 cells were incubated with PBS, 0.1 mM MGO, 0.3 mM MGO or 1 mM MGO for 4 hours. A. Micrographs of typical PC12 cells. B. Tryphan blue staining of PC12 cells. Bars represent three independent experiments carried out in quadruplicates. Cell viability of cells cultured in the presence of PBS ( = control) was set to 1 and cell viability expressed in relation to the control. C. FACS analysis of PC12 cells stained with annexin V and propidium iodide.</p

AGE-modification of PC12 cells using MGO.

<p>PC12 cells were incubated with PBS, 0.1 mM MGO, 0.3 mM MGO or 1.0 mM MGO for 4 hours. A. Washed cells were solubilized and subjected to SDS-gel electrophoresis. Proteins were blotted and detected using monoclonal CML26 antibody B&C. Permeabilized (B) and non-permeabilized (C) were analyzed by flow cytometry using monoclonal CML26 antibody.</p

Real-time analysis of neurite outgrowth of AGE-modified PC12 cells.

<p>PC12 cells were AGE-modified using 1 mM MGO for 4 h as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g004" target="_blank">Figs. 4</a> &<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g005" target="_blank">5</a>. Cells were cultured on LN-coated E-plates and neurite outgrowth was induced by application of 100 ng/ml NGF. Neurite outgrowth was continuously quantified over 48 hours by RTCA as described in Pollscheit et al. (2012). Total neurite outgrowth of non-modified control cells during 48 h was set to 100% and neurite outgrowth of AGE-modified cells was expressed in % of control. Bars represent two independent experiments carried out in quadruplicates.</p

Cell adhesion of PC12 cells to AGE-modified ECM proteins.

<p>AGE-modified and non-modified collagen IV and laminin (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112115#pone-0112115-g002" target="_blank">Fig. 2</a>) were coated on E-plates. Cell adhesion of 5×10⁵ PC12 cells was quantified by RTCA real time analysis as described. Adhesion to non-modified ( = control) substrates was set to 100% and adhesion to AGE-modified substrates was calculated in % of control. Each bar represents values of three independent experiments carried out in triplicates (*p≤0.0001).</p

Formation of advanced glycation endproducts (AGE).

<p>Formation of advanced glycation endproducts (AGE).</p

AGE modification of ECM proteins using MGO.

<p>20 µg collagen IV (Col. IV) or laminin (LN) were spotted on a nitrocellulose membrane. One half of the membrane was incubated with 1 mM MGO for 4 hours. AGE formation was detected by dot blot analysis using the monoclonal CML26 antibody.</p

Relative mRNA accumulation of genes involved in dicarbonyl defence and hypoxia.

<p>Relative mRNA accumulation of genes involved in dicarbonyl defence and hypoxia.</p

Kinase phosphorylation in MCF-7-Md and TamR-Md cells in response to glyoxal and methylglyoxal.

<p>Confluent and serum starved cells were incubated with different concentrations of dicarbonyls for 10 minutes before proteins were extracted and subjected to Western blot analysis. Experiments were repeated three times in duplicate. A representative Western blot and the quantification of the signals of all experiments are shown. Band intensity was expressed relative to the β-actin signal and control treatments were set to 1 for each cell-line.</p

Phosphorylation of kinases in MCF-7-Hd and –Dk and TamR-Hd and -Dk cell lines in response to dicarbonyls.

<p>7A: MCF-7-Hd and TamR-Hd cell lines 7B: MCF-7-Dk and TamR-HDk cell lines. Cells were treated as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101473#pone-0101473-g005" target="_blank">figure 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101473#pone-0101473-g006" target="_blank">6</a>. Experiments were performed at least three times in duplicate and the result of one representative experiment is shown.</p

AGE accumulation under exogenous aldehyde stress.

<p>A: AGE accumulation (CML (left) and MG-AGE (right)) in MCF-7-Md (lower panel) and TamR-Md cells (upper panel) after cultivation for three days with different concentrations of methylglyoxal and glyoxal as shown by Western blotting. Strongly enhanced AGE accumulation became visible when cell number was significantly reduced due to toxic effects, as represented by the decreasing β-actin signal. Glyoxal resulted in accumulation of CML whereas methylglyoxal treatment caused MG-AGE modification. Cells were treated as described for the determination of vitality/proliferation. Proteins were extracted per well and not corrected for protein amount. Therefore, the blots represent adherent cells only. B) AGE accumulation shown by immunofluorescence. Composite images of dual exposures are shown. MCF-7-Md cells were stained for CML and MG-AGE (red) by specific antibodies as indicated and nuclear staining was achieved with DAPI (blue) of cells treated for 24 h with 1 mM methylglyoxal or glyoxal.</p