Effects of the combination of space radiation and darkness.

<p>Polµ (<b>A</b>), DNA-PKc (<b>B</b>) and Gadd45 (<b>C</b>) mRNAs were quantified by qPCR in larvae that developed in the dark and under the simulation of ISS radiation. Polµ proteins were quantified by western blotting (<b>D</b>). Each lane contains proteins from 3 larvae. Western blot results were analyzed and quantified by densitometry using a Fusion Fx7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) and Polµ expression was normalized to β-actin (<b>E</b>). Results are expressed as the mean ± SEM. “A.U.” stands for “arbitrary units”.</p

Effects of spaceflight conditions.

<p>Polµ mRNAs were quantified in larvae that developed in microgravity in the ISS (<b>A</b>), in hypergravity (<b>B</b>), in the RPM to simulate microgravity (<b>C</b>) or in the dark (<b>D</b>). The same study was performed with larvae subjected to a mechanical stress (<b>E</b>) or the ISS radiation environment (<b>F</b>). The effects of space radiation on DNA-PKc (<b>G</b>) and Gadd45 (<b>H</b>) mRNAs and on the amount of Polµ protein (<b>I</b> and <b>J</b>) were also investigated. Western blot results were analyzed and quantified by densitometry using a Fusion Fx7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) and Polµ expression was normalized to β-actin (<b>J</b>). Each lane of the western blot contains proteins from 3 larvae. Results are expressed as the mean ± SEM. “A.U.” stands for “arbitrary units”.</p

Primers used in this study.

<p>Primers used in this study.</p

<i>Pleurodeles waltl</i> DNA polymerase µ.

<p>Schematic representation of <i>P. waltl</i> Polµ mRNAs (<b>A</b>). The short variant (Polµ Δ Ex 8) presents a 103 bp deletion corresponding to exon 8. This deletion removes one part of the active site of the protein. The BRCT (breast cancer suppressor protein carboxy-terminal) and the Pol X domain, which contains the active site, are required for Polµ function. Polµ variants were amplified using primers indicated by horizontal arrows (<b>B</b>). Quantification of the obtained PCR products using a Gel Doc 2000 and the Quantity One v.4.3.1 software (Bio-Rad, Hercules, CA, USA) indicates that the short isoform represents 16% and 6% of the Polµ transcripts in the spleen and testis of <i>P. waltl</i>, respectively. Ctl⁻ = negative control. MW = 100 bp DNA ladder. A cladogram was then constructed by neighbor joining (<b>C</b>) supported with 1000 bootstrap replications using the MEGA4 software (<a href="http://www.megasoftware.net/" target="_blank">http://www.megasoftware.net/</a>) and the following sequences: Terminal deoxynucleotidyl transferase (TdT); <i>Bos taurus</i> (DAA14763), <i>Homo sapiens</i> (BAB72001), <i>Mus musculus</i> (NP_001036693), <i>Gallus gallus</i> (NP_990720), <i>Ambystoma mexicanum</i> (AA092254), <i>Xenopus laevis</i> (NP_001079251), <i>Danio rerio</i> (AAS89780). Polymerase beta; <i>Mus musculus</i> (NP_035260), <i>Homo sapiens</i> (NP_002681), <i>Xenopus laevis</i> (NP_001081643), <i>Xenopus tropicalis</i> (AAH74537), <i>Xiphophorus maculatus</i> (AAU11319). Polymerase lambda; <i>Danio rerio</i> (NP_998408), <i>Xenopus tropicalis</i> (NP_001093716), <i>Mus musculus</i> (NP_064416), <i>Homo sapiens</i> (NP_001167555). Polymerase mu; <i>Homo sapiens</i> (NP_037416), <i>Mus musculus</i> (NP_059097), <i>Danio rerio</i> (NP_956542), <i>Xenopus tropicalis</i> (NP_001164987), <i>Pleurodeles waltl</i> (HE583591). The scale bar corresponds to the evolutionary distance.</p

Protein oxidation evaluated by measuring the carbonylation of amino acid side chains.

<p>Representative oxiblots of carbonylated proteins from larvae submitted to space radiation (<b>A</b>) or larvae that developed in the dark and under the simulation of ISS radiation (<b>B</b>) are shown. Each lane contains proteins from 3 larvae. Equal protein loading was assessed by Coomassie staining. Western blot results were analyzed and quantified by densitometry using a Fusion Fx7 imaging system (Vilber Lourmat, Marne-la-Vallée, France) and carbonylation was normalized to the total amount of protein. Results are expressed as the mean ± SEM.</p

Body weight gain, lymphoid organ normalized weight and spleen cellularity in control (C), restrained (R) and hindlimb unloaded (HU) mice at the end of the experiments.

<p>C  =  Control, R  =  Restrained, HU  =  Hindlimb Unloaded.</p><p>* <i>p</i> = 0.032 <i>versus</i> R, ^¤<i>p</i> = 0.002 <i>versus</i> C.</p><p>Body weight gain was calculated with the formula [body weight on day 21 - body weight on day 0 of the treatment]. Normalized weights were calculated with the ratio [organ weight/body weight] for each mouse. The number of splenic nucleated cells was calculated after red blood cell lysis with NH₄Cl. Each group was compared to the others. <i>n</i> = 5 mice per group. Differences were found to be statistically significant using an ANOVA and Tukey <i>post-hoc</i> test. HU mice did not gain weight (<i>p</i> = 0.032 <i>versus</i> R, <i>p</i> = 0.002 <i>versus</i> C), while the other groups grew by approximately 7% (R) to 11% (C) in comparison to their initial weight. No significant difference was found between the three experimental groups for lymphoid organ normalized weights. The number of nucleated cells was reduced by 19% and 25%, respectively, in the R and HU mice in comparison to C mice, although these differences were not significant (<i>versus</i> R: <i>p</i> = 0.479; <i>versus</i> HU: <i>p</i> = 0.273). Data are mean values ± SEM.</p

Serum corticosterone concentration in control (C), restrained (R) and hindlimb unloaded (HU) mice.

<p>Trunk blood was collected immediately after the sacrifice. Corticosterone concentration was measured using an ELISA kit with a detection threshold of 16.9/ml. Each experimental group was compared to the others. <i>n</i> = 4, 6 and 4 mice for C, R and HU groups, respectively. No significant difference was found using an ANOVA and Tukey <i>post-hoc</i> test.</p

Absolute numbers of lymphocytes and lymphocytes subsets in the spleen (x10⁶).

<p>C  =  Control, R  =  Restrained, HU  =  Hindlimb Unloaded.</p><p>* <i>p</i> = 0.039 <i>versus</i> C; ^¤<i>p</i> = 0.011 <i>versus</i> C.</p><p>Absolute numbers for each cell population was calculated using the number of splenic nucleated cells and the percentages obtained by flow cytometry. Each group was compared to the others. <i>n</i> = 4, 5 and 4 mice for C, R and HU groups, respectively. Differences were found to be statistically significant using an ANOVA and Tukey <i>post-hoc</i> test. The number of total lymphocytes was reduced by 25% and 33%, respectively, in R and HU mice in comparison to C mice. There was no significant difference for T cells and their subpopulations for the three groups. The number of B cells was significantly decreased by 44% (<i>p</i> = 0.039) and 59% (<i>p</i> = 0.011) in R and HU mice, respectively. Data are mean values ± SEM.</p

Phenotypes of splenic lymphocytes from control (C), restrained (R) and hindlimb unloaded (HU) mice after 72 h of incubation without mitogen.

<p>After incubation with fluorescent antibodies, cell populations were identified by flow cytometry. Lymphocytes were first gated according to their size and granularity. Populations of interest were then expressed as percentages of the lymphocyte population. Each experimental group was compared to the others. <i>n</i> = 5 mice per group. No significant difference was found using an ANOVA and Tukey <i>post-hoc</i> test.</p

Detection of lymphocyte division after 72<i>in vitro</i> stimulation with or without mitogen.

<p>Splenocytes were labeled with CFSE and cultured for 72(LPS or ConA). For each mouse, dividing lymphocytes were determined as in the three examples presented here. (<b>A</b>) Cells were labeled with fluorescent antibodies and analyzed by flow cytometry. They were sorted with FSC/SSC profiles, separating viable lymphocytes (black arrow) from dead cells and cellular debris. (<b>B</b>) Among the viable lymphocyte gate, each subpopulation was selected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092664#pone-0092664-g003" target="_blank">Figure 3</a> and assessed for fluorescent division peaks. Generation 0 (black arrow) and each of the following generations (pink area) were used by the software to calculate the number of dividing cells.</p