List of biological processes of the 30 proteins analyzed.

<p>List of biological processes of the 30 proteins analyzed.</p

Vitamin C alters the amount of specific endoplasmic reticulum associated proteins involved in lipid metabolism in the liver of mice synthesizing a nonfunctional Werner syndrome (Wrn) mutant protein

<div><p>Werner syndrome (WS) is a premature aging disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domain. Mice lacking the helicase domain of the Wrn protein orthologue exhibit transcriptomic and metabolic alterations, some of which are reversed by vitamin C. Recent studies on these animals indicated that the mutant protein is associated with enriched endoplasmic reticulum (ER) fractions of tissues resulting in an ER stress response. In this study, we identified proteins that exhibit actual level differences in the ER enriched fraction between the liver of wild type and Wrn mutant mice using quantitative proteomic profiling with label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Multiple Reaction Monitoring (MRM) and immunoblotting were performed to validate findings in a secondary independent cohort of wild type and Wrn mutant mice. DAVID 6.7 (NIH) was used for functional annotation analysis and indicated that the identified proteins exhibiting level changes between untreated wild type, Wrn mutant, and vitamin C treated Wrn mutant mice (ANOVA <i>P</i>–value < 0.05) were involved in fatty acid and steroid metabolism pathways (Bonferroni <i>P</i>-value = 0.0137). Finally, when we compared the transcriptomic and the proteomic data of our mouse cohorts only ~7% of the altered mRNA profiles encoding for ER gene products were consistent with their corresponding protein profiles measured by the label-free quantification methods. These results suggest that a great number of ER gene products are regulated at the post-transcriptional level in the liver of Wrn mutant mice exhibiting an ER stress response.</p></div

Influence of a pre-stimulation with chronic low-dose UVB on stress response mechanisms in human skin fibroblasts

<div><p>Exposure to solar ultraviolet type B (UVB), through the induction of cyclobutane pyrimidine dimer (CPD), is the major risk factor for cutaneous cancer. Cells respond to UV-induced CPD by triggering the DNA damage response (DDR) responsible for signaling DNA repair, programmed cell death and cell cycle arrest. Underlying mechanisms implicated in the DDR have been extensively studied using single acute UVB irradiation. However, little is known concerning the consequences of chronic low-dose of UVB (CLUV) on the DDR. Thus, we have investigated the effect of a CLUV pre-stimulation on the different stress response pathways. We found that CLUV pre-stimulation enhances CPD repair capacity and leads to a cell cycle delay but leave residual unrepaired CPD. We further analyzed the consequence of the CLUV regimen on general gene and protein expression. We found that CLUV treatment influences biological processes related to the response to stress at the transcriptomic and proteomic levels. This overview study represents the first demonstration that human cells respond to chronic UV irradiation by modulating their genotoxic stress response mechanisms.</p></div

CPD repair rate is enhanced by the CLUV pre-stimulation in NHDF.

<p><b>(A)</b> Immuno-slot-blot showing the level of CPD and DNA. NHDF were irradiated with a single UVB dose (Acute), a CLUV irradiation or a CLUV followed by a single acute UVB dose (CLUV+Acute), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173740#pone.0173740.g001" target="_blank">Fig 1</a>. Zero and 24 h post-irradiation, DNA was harvested and applied on a membrane. Revelation of CPD and DNA was performed using specific monoclonal antibodies. NoUV is used as a negative control and DNA as a loading control. <b>(B)</b> Representation of the quantity of CPD repair after different UVB treatments. Quantitative analysis of the immuno-slot-blot detecting CPD is performed by measuring the signal intensity at each time points post-UV and compare it to the 0 h for each UVB treatment condition (Acute, CLUV, CLUV+Acute). The signal at 0 h corresponds to 100% of the CPD signal for each UVB treatment independently. The results then describe the CPD removal relative to the initial CPD amount for each UVB treatment (Acute, CLUV and CLUV+Acute). Normalization is performed using the corresponding DNA signal as previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173740#pone.0173740.ref031" target="_blank">31</a>]. Results are presented as means ± SEM. <i>P-value</i> was evaluated using the student’s <i>t-test (</i>*<i>p</i> < 0.05; <i>*</i>*<i>p</i> < 0.01). Experiment was performed using 3 strain cells (N = 3) at least in duplicate (n = 2).</p

List of biological processes deregulated by a CLUV treatment.

<p>List of biological processes deregulated by a CLUV treatment.</p

CLUV treatment induces proteomic changes.

<p>Three cell strains of NHDF subjected or not to a CLUV treatment and proteins were extracted. Proteins from the triplicate were pooled and proteome change was analyzed. <b>(A)</b> 2D-DIGE depicting protein expression differences between CLUV and untreated NHDF. The control (NoUV) was labeled with cy3 (left panel) and CLUV-treated cells (CLUV) with cy5 (middle panel). After labeling, proteins were separated on a 2D-DIGE according to their molecular weight and pH. Gels were merged (cy3/cy5) (right panel) to see proteomics changes. <b>(B)</b> Merged 2D-DIGE gel (cy3/cy5) depicting proteomic changes. Proteins with equal abundance between the CLUV-treated and the untreated NHDF are shown in yellow spot, while up-regulated proteins by the CLUV treatment appears in red and down-regulated in green. Full circles display up-regulated protein and dashed circles exhibit down-regulated proteins. A total of 30 proteins were further analyzed by mass spectrometry.</p

Protein levels of GRP78 and HSC70 proteins in the ER enriched fractions from mice.

<p>(A) Example of western blots showing protein levels of GRP78, HSC70, and calreticulin in liver ER enriched fractions of mice. (B) Ratio of GRP78 signal over calreticulin signal from the western blots. (C) Ratio of HSC70 signal over calreticulin signal from the western blots. (All Tukey post ANOVA and <i>t</i>-tests <i>P</i>-values > 0.05). Bars in all histograms represent SEM of four mice.</p

Schematic representation of the irradiation protocol.

<p>Confluent NHDF were irradiated with different UVB irradiation protocols. Three conditions were used: (1) single UVB dose, (2) CLUV treatment and (3) CLUV followed by a single UVB dose. (1) Acute treatment is a single UVB irradiation of 400 J/m²; (2) CLUV treatment consists UVB irradiations of 75 J/m² every 12 h for 7.5 days (15 irradiations). (3) Cells are irradiated with the CLUV treatment described in (2) followed by the single UVB irradiation described in (1) 12 h after the last CLUV irradiation. Cells subjected to the different irradiation protocols were then used for further analysis.</p

Efficiency of the liver ER enriched fractionation procedure on the different groups of mice.

<p>(A) Schematic representation of the different steps undertaken to obtain different cellular fractions. (B) Example of western blots showing protein levels of GRP78, HSC70, calreticulin, MnSOD, catalase, topoisomerase I, and SVCT1 in the different liver fractions. Each lane contains 15 μg of proteins. (H = whole cell homogenate; P = pellet fraction from the first step of the procedure; S = supernatant fraction of the last step of the procedure; ER = ER enriched fraction). (C) Principal component analysis (PCA) graph showing the consistency of the fractionation procedure when we measured the protein levels from the immunoblots shown in Fig 2B in the different fractions of vitamin C treated and untreated <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice compared to wild type mice. WT = wild type mice; Wrn = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice; Wrn+VitC = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice treated with 0.4% vitamin C (w/v) in drinking water since weaning. X and Y axis show principal component 1 and principal component 2 that explain 48.7% and 33.6% of the total variance, respectively. Prediction ellipses are such that a new observation from the same group will fall inside the ellipse with a probability of 0.95.</p

Schematic representation of the different steps undertaken to obtain a list of protein differentially expressed between our different mouse cohorts at a statistical significant level.

<p>The number of proteins identified in each step is indicated in parentheses. WT = wild type mice; Wrn = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice; Wrn+VitC = <i>Wrn</i>^{<i>Δhel/Δhel</i>} mice treated with 0.4% vitamin C (w/v) in drinking water since weaning.</p