To test these hypotheses, several experimental approaches have been performed in thymocytes from male and female C57BL/6J mice (Fig. 1). First, caspase-3 amounts were estimated by Western blot to analyze the effect of administering priming low dose radiation prior to a high dose, as well as exposure to a single low or high dose of radiation, on the level of apoptosis. Second, we investigated by cDNA microarray analysis the transcript profiles of 1944 genes involved in apoptosis signaling (included in the Agilent Mouse Gene Expression G3 8x60K microarray) according to GO-Ontology database under the mentioned experimental conditions. Third, quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was used to validate significant data from cDNA microarray analysis. Finally, protein amounts of phosphorylated forms of TRP53 at serine 18 and serine 389 were determined by Western blot in response to the adaptive regimen of radiation and after exposure to single low or high dose of radiation.
Mice and irradiation
Male and female C57BL/6J mice were purchased from the Charles River Laboratories and kept 1 week in the local animal house for acclimatization. Animal experiments were carried out according to the European Commission Guidelines (Directive 86/609/CEE) on the use of laboratory animals.
Three groups of Irradiated males and females of comparable age (4 to 5 weeks) and weight (21–24 g) were used in this study: group subjected to an adaptive regimen of radiation (RAR group), low dose exposed group and group subjected to high dose exposure. Each experimental group consisted of three individuals. Mice from RAR group were irradiated with a priming dose of 0.075 Gy of X-rays, that it is known to known to induce an effective in vivo radiaodaptive response in mouse thymocytes [21], generated with a Philips MCN 101 X-ray generator operating at 100 kV/15 mA with 1 mm of Be and 3 mm AI added filtration. With an interval of 6 h, that it has been used for radio-adaptive studies with thymocytes [21], these mice were further irradiated with challenging dose of 1.75 Gy of γ-rays that were generated by a 137Cs gamma IBL-437C irradiator (CIS bio international, Gif-sur-Yvette, France). Mice from low dose irradiation group were exposed to a single dose of X-rays (0.075 Gy). Mice from high dose irradiation group were treated with 1.75 Gy of γ-rays alone. Control group consisted of non-irradiated mice.
Thymic cell fractionation
Mouse thymus samples were mechanically dispersed and strained through a Nylon Mesh Cell Strainer of 40 μM (BD Biosciences, San Jose, CA) to isolate the thymocytes.
Apoptosis analysis
Since pro-caspase 3 is cleaved into the 17–19 kDa and 12 kDa subunits only when cells undergo apoptosis, we used the fragmentation of caspase 3 as indicator of apoptosis induction. Cleaved caspase 3 (17/19 kDa) were determined by Western blotting and assessed by densitometry analysis.
Transcriptome analysis
cDNA microarray experiments were performed using the Agilent Mouse Gene Expression G3 8x60K chip (Agilent Technologies, Palo Alto, CA). Three independent thymocyte samples for each experimental condition were used in this analysis. Total RNA was extracted by combination of Trizol reagent (Invitrogen, Carlsbad, CA), MaXtract high density extraction column (Qiagen, Hilden, Germany) and purification using the RNeasy Mini kit (Qiagen). Following extraction, total RNA were checked for RNA integrity (Agilent 2100 Bioanalyzer, Agilent Technologies). All samples showed common high quality RNA Integrity Numbers (RIN 9.0–9.6) and RNA was quantified by photometric measurement using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). A pool of amplified RNAs obtained from the Universal Mouse RNA (Stratagene, La Jolla, CA) was used as a reference. Briefly, 100 ng of total RNA was converted to cDNA, followed by in vitro transcription and incorporation of Cy5-dCTP (test) and Cy3-dCTP (reference) into nascent cRNA. After fragmentation, labeled cRNA was hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarrays for 17 h at 65 °C. Quality control parameters of cRNA labeling and hybridization performance were found within the manufacturers specifications. Arrays were scanned as described by the manufacturer. Signal intensities on 20 bit tiff images were calculated by Feature Extraction software (FE, Vers. 10.7.1.1; Agilent Technologies, Palo Alto, CA). Normalized signal values were then obtained by Cy5/Cy3 ratio computing and logarithmic base 2 transformations with the GeneSpring GX12 software (Agilent). Before statistical analysis, a new quality control was performed to filter out questionable and outlier expression values. Anyone normalized expression value across the samples in each experimental condition that was further away from the average, was ruled out considering it as an outlier.
GO Ontology analysis
In this study, we analyzed only those genes related with apoptosis included in the mentioned cDNA microarray (see Additional file 1: Table S1). For this purpose, we selected genes that almost one of the GO Ontology identifiers associated in the Agilent microarray annotation data was included in a list of 387 GO-IDs related with apoptosis in the Gene Ontology database of the annotation data package “GO.db” (GO.db: A set of annotation maps describing the entire Gene Ontology. R package version 3.1.2. Carlson M. http://bioconductor.org). This list of apoptosis GO-IDs was created from the total number of GO identifiers registered in this database (38027), selecting those GO-Terms-Name (TERM) or GO-Term-Definition (DEF) included the words “apoptosis and/or apoptotic process” (Additional file 2: Table S2).
Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Extracted RNAs were then quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Reverse transcription was performed using 1 μg of total RNA for cDNA synthesis with the High Capacity RNA-to-cDNA reverse transcription kit (Applied Biosystems, Foster City CA, USA) using random oligonucleotide primers. All the quantitative real-time PCR were carried out in 10 μl volume on ABI 7900HT Real-Time PCR system (Applied Biosystems, Carlsbad, CA) into 384-well plates using GoTaq qPCR Master Mix (Promega, Madison, WI). Amplicons were designed to span intron-exon boundaries. Primer efficiencies were calculated prior to experimental use and amplification efficiencies were greater than 90 % for all primer sets. Amplifications using specific primers (Additional file 3: Table S3) were done with a denaturation step at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 3 s and primer annealing at 59 °C for 30 s. Upon completion of the cycling steps, a final step at 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s was done and then the reaction was stored at 4 °C. Reactions were run in triplicate in three independent experiments. The geometric mean of housekeeping genes HPRT1 and PPIA were used as an internal control to normalize the variability in expression levels and were analyzed using the 2-ΔΔCT method described elsewhere [25].
Western blot analysis
Isolated thymocytes were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with PhosStop phosphatase inhibitor and Complete EDTA free protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany). The concentrations of thymocytes proteins were measured using a Pierce BCA Protein assay kit (ThermoFisher Scientific, Wilmington, DE) and a Benchmark Microplate Reader (Bio-Rad, Hercules, CA). Proteins (5 μg) were mixed in equal volume with 2× sample buffer (0.125 mol/l Tris-HCl, 4 % SDS, 20 % glycerol, 10 % 2-mercaptoethanol, 0.002 % bromophenol blue, pH 6.8). These samples were boiled at 99 °C for 5 min, and immediately cooled on ice. Electrophoresis was performed using 4–15 % Mini-PROTEAN TGX™ precast gels (BioRad, Hercules, CA). The proteins of electrophoresed gels were transferred to a polyvinylidene difluoride membrane (PVDF) (Millipore, Temecula, CA). The sizes of proteins were confirmed with the Precision Plus Protein Dual Color Standards (Bio-Rad).
After blocking, membranes were incubated with each primary antibody in either 5 % w/v BSA or nonfat dry milk, 1× TBS, 0.1 % Tween 20 overnight at 4 °C. As primary antibodies, p53 (1C12) (#2524; Cell Signaling), caspase-3 (Asp175) (#9661; Cell Signaling), and phospho-p38MAPK (Thr180/Tyr182) (#9211; Cell Signaling) were used at 1:1000 dilution. For β-actin (A 5316; Sigma) was used a dilution of 1:5000. Phospho-p53 (Ser15) (#9284; Cell Signaling) and phospho-p53 (Ser392) (#9281; Cell Signaling) were used at 1:500. Membranes were washed with TBST buffer and incubated for 60 min at room temperature with secondary antibodies we used Anti-rabbit IgG, HRP-linked Antibody (#7074; Cell Signaling) and Anti-mouse IgG, HRP-linked Antibody (#7076; Cell Signaling) at 1:1000 dilution. Bound antibodies were visualized by chemiluminescence using WesternBright™ ECL detection kit reagent (Advansta, Menlo Park, CA). Luminescent images were analyzed using ImageQuant LAS 4000 biomolecular imager analyzer (GE Healhcare, Buckinghamshire, UK). For densitometry analysis Scion Image software (Scion Corporation) analyzer program was used.
Statistical analysis
cDNA microarray data analysis
To find sex differences in the transcriptional expression of genes involved in apoptosis signaling that are influenced by RAR, male and female transcriptional expression were compared under all experimental conditions by performing the statistical contrasts as follows: contrast 1 (male RAR vs female RAR); contrast 2 (male RAR - female RAR) vs (male control - female control); contrast 3(male RAR - female RAR) vs (male high dose irradiation - female high dose irradiation); and contrast 4 (male RAR - female RAR) vs (male low dose irradiation - female low dose irradiation). For those genes differentially expressed in all the contrasts, the difference between their expressions in male and female in RAR radiation condition is significant from any other difference found in any other radiation or control condition. These genes were selected as endpoints of this analysis.
The log-transformed (base 2) normalized values of expression from cDNA microarray experiments were used as the source of all raw data for statistical analysis in this section, and to detect differentially expressed genes in the contrasts described before, moderated t-test analysis was conducted with functions of the limma Package (R statistical software). P values were adjusted by the Benjamini-Hochberg method to control false discovery rate (FDR). And to consider a gene as differentially expressed, results from moderated t-test should show a FDR <0.05, and there should be an expression fold-change (FC) ≥1.5.
Additionally, to assess sex differences in gene expression taking into account at the same time the effect of the radiation treatment used, a two way ANOVA analysis was also applied.
Analysis of RT-PCR data
Mean expression values of selected genes were also calculated by quantitative real-time RT-PCR between RAR and the control condition in each sex, and compared using t-tests.
Analysis of protein expression data
To assess the sex differences across the experimental groups in protein amounts detected by Western blot analyses, multivariate linear regression models were performed including normalized mean values obtained from three independent experiments as the outcome, and sex, type of radiation treatment and an interaction term between them as independent variables.
Correlation coefficient analysis
Pearson product-moment correlation coefficients and their statistical significance were calculated to assess the strength and the direction of the associations between expressions.
All statistical analyses were performed using R Software (R Core Team (2013). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/).