Immunoblot and RPPA analysis of the senescence-associated proteins p21 and members of PI3K/Akt/mTOR pathway.

<p>A representative immunoblot of p21 expression levels at different time points and dose rates is shown (A). The columns represent protein levels of p21 (B) Akt (C), phospho-Akt (D), PI3K (E) and mTOR (F) in control (blue), 1.4 mGy/h (orange) and 2.4 mGy/h (green) irradiated HUVECs. The average ratios of relative protein expression in control and irradiated samples are shown. The protein bands were quantified using TotalLab TL100 software by integration of all the pixel values in the band area after background correction and normalized to the actin expression. The data are represented as ± SEM. Three biological replicates were used in all experiments. (Students t-test; *p<0.05, **p<0.01 and ***p<0.005).</p

List of selected antibodies analyzed using RPPA.

<p>List of selected antibodies analyzed using RPPA.</p

Schematic representation of the ICPL-triplex proteomic approach work flow.

<p>The protein samples from week 10 (control and two irradiated) were reduced and alkylated before labeling with ICPL0, ICPl4 and ICPL6. Samples were mixed and further separated using 1D gel electrophoresis and digested as described in Methods. Samples were analyzed by LC-ESI-MS/MS. Quantification of proteins was performed by Proteome Discoverer software using three biological replicates.</p

Canonical pathways of deregulated proteins.

<p>Canonical pathways of deregulated proteins.</p

Schematic representation of pathways altered by chronic low-dose-rate radiation in HUVECs.

<p>Orange and green colors represent down-regulated proteins detected by RPPA and ICPL triplex proteomic methods, respectively. Blue color indicates unregulated proteins detected by the RPPA technique.</p

Graphical representation of deregulated proteins representing a merge of two most affected functional networks at week 10 (2.4 mGy/h).

<p>Differentially up- or down-regulated proteins are marked in red and green, respectively. Nodal molecules Akt and ERK 1/2 (blue color) were predicted by the IPA software as central transcriptional regulators.</p

RPPA analysis of senescence-associated proteins.

<p>The columns represent protein levels of phospho to total ERK ½ (A), Rho GDI (B), caldesmon (C), nucleolin (D) and SUMO1 (E) in control (blue), 1.4 mGy/h (orange) and 2.4 mGy/h (green) irradiated HUVECs. The average ratios of relative protein expression in control and irradiated samples are shown. The data are represented as ± SEM. Three biological replicates were used in all experiments. (Students t-test; *p<0.05, **p<0.01 and ***p<0.005).</p

Growth curve and senescence associated β-gal of HUVECs exposed to chronic low-dose rates.

<p>A) The growth curve is plotted with cumulative population doublings versus time. Growth curves of control (blue), 1.4 mGy/h (orange) and 2.4 mGy/h (green) irradiated HUVECs are shown. Cumulative population doublings from each week are presented as means ± SEM (n = 3). B) Histograms of positively-stained cells for SA-ß-gal (senescence marker) for control (blue), irradiated by 1.4 mGy/h (orange) and irradiated by 2.4 mGy/h (green). Data are presented as means ± SEM (n = 3). (Students t-test; *p<0.05, **p<0.01 and ***p<0.005).</p

PPAR Alpha: A Novel Radiation Target in Locally Exposed <i>Mus musculus</i> Heart Revealed by Quantitative Proteomics

Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation

PPAR Alpha: A Novel Radiation Target in Locally Exposed <i>Mus musculus</i> Heart Revealed by Quantitative Proteomics

Radiation exposure of the thorax is associated with a markedly increased risk of cardiac morbidity and mortality with a latency period of decades. Although many studies have confirmed the damaging effect of ionizing radiation on the myocardium and cardiac endothelial structure and function, the molecular mechanism behind this damage is not yet elucidated. Peroxisome proliferator-activated receptor alpha (PPAR alpha), a transcriptional regulator of lipid metabolism in heart tissue, has recently received great attention in the development of cardiovascular disease. The goal of this study was to investigate radiation-induced cardiac damage in general and the role of PPAR alpha in this process in particular. C57BL/6 mice received local heart irradiation with X-ray doses of 8 and 16 gray (Gy) at the age of 8 weeks. The mice were sacrificed 16 weeks later. Radiation-induced changes in the cardiac proteome were quantified using the Isotope Coded Protein Label (ICPL) method followed by mass spectrometry and software analysis. Significant alterations were observed in proteins involved in lipid metabolism and oxidative phosphorylation. Ionizing radiation markedly changed the phosphorylation and ubiquitination status of PPAR alpha. This was reflected as decreased expression of its target genes involved in energy metabolism and mitochondrial respiratory chain confirming the proteomics data. This study suggests that persistent alteration of cardiac metabolism due to impaired PPAR alpha activity contributes to the heart pathology after radiation