Peroxidase activity.

*<p>The amount of t-BHP reduced was calculated from the amount NADPH oxidized after 6 min. The mixture contained 50 µM GSH, 0.2 mM NADPH, 2 mM EDTA, 0.1 mg/mL BSA, and 6 µg/mL yGR and 1 µM Grx in 100 mM Tris-HCl. Selenite and SAM were added to a final concentration of 5 µM and 4 mM respectively.</p

Inhibition of thioredoxin mediated protein disulfide reductase activity.

<p>The method was performed as described by Kumar et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050727#pone.0050727-Kumar1" target="_blank">[6]</a>. The reaction mixture contained 80 mM HEPES buffer, pH 7.6, 3 mM EDTA and 0.7 mM NADPH. TrxR1 and Trx1 were added to a final concentration of 8 nM and 1 µM respectively. The measurements were performed with the following selenite concentrations (1, 5 and 10 µM). The amount of SH-group formed was measured at 412 nm. Grey bars: Addition of selenite at varying concentrations. White bars: Addition of both selenite and SAM (4 mM). Student t-test, dependent by samples, was used for statistical analysis (*p<0.05, **p<0.01).</p

Effect of selenium compounds on superoxide production.

<p>H157 cells were stained with hydroethidine and treated for 5 h with selenite (5 µM) +/− SAM (500 µM), and MSA (5 µM) before detection of accumulated superoxide produced by FACS analysis, as described under materials and methods.</p

A–D. Total selenium accumulation, extracellular and intracellular thiols after treatment with various selenium compounds.

<p>Selenium accumulation in ng/mg protein after 5 h treatment with A) 5 µM selenite +/− SAM and MSG B) 5 µM GS-Se-SG +/− SAM and MSG measured by GF-AAS analysis. C) Total extracellular thiol content after 5 h treatment with 5 µM selenite +/− SAM and MSG. (*p<0.05 compared to control) and D) total intracellular thiol content following 5 h treatment with selenite (5 µM) +/− SAM was determined by the DTNB assay. Statistical analysis was performed by one-way ANOVA (95% confidence interval) followed by Tukey-Kramer multiple comparison test. (*p<0.05, **p<0.01 and ***p<0.001 compared to controls, °p<0.01 compared to selenium treated cells).</p

Additional file 4: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Detailed candidate RFX regulator oPOSSUM3 scanning results using JASPAR 2016 core vertebrate TF binding profiles. Transcription factor binding sites (TFBS) scanning results from oPOSSUM3 within the promoter and enhancer regions of RFX1–8 using the CORE vertebrate TF binding profiles in JASPAR 2016. Included are the DNA regions that were considered as foreground and the following TF binding site details: SP2 (specificity protein 2) (JASPAR profile MA0516.1) and ESR1 (estrogen receptor alpha) (MA0112.3). (XLSX 50 kb

Additional file 5: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Ct levels of qRT-PCR, used for validation of candidate RFX regulators by siRNA knockdown. Individual Ct levels with automatic threshold obtained on an AB7500 Fast machine for SP2 and ESR1 as candidate RFX regulators and their respective test siRNA and scrambled (Scr) control siRNA knockdown data on RFX genes (RFX1, RFX2, RFX3, RFX5, RFX7) and the two reference genes (HPRT1, HSPCB). (XLSX 33 kb

Peroxidase activity.

*<p>The amount of t-BHP reduced was calculated from the amount NADPH oxidized after 6 min. All experiments were performed in TE-buffer with human Trx (2 µM), TrxR (50 nM), and NADPH (500 µM). Final concentration of selenite was 5 µM and 4 mM for SAM.</p

Additional file 2: of Characterization of the human RFX transcription factor family by regulatory and target gene analysis

Hierarchical clustering, expression plots and top 10 tissues, primary cells and cell lines of RFX TSS locations. Hierarchical clustering of 30 RFX TSS locations (with shorthand p for promoter) based on expression values (TPM) across 135 human tissue samples, using a 1-Pearson correlation distance measure and average linkage method, as computed by the pvclust R package with nboot = 1000 with the numbers representing approximately unbiased (au) p-values (Suzuki and Shimodaira, 2006). Tissue clusters are color-coded and represent the groups of tissues with the highest overall expression values: immune system (teal), gastrointestinal tract (purple), testis (green), brain and spinal cord (red), and two minor clusters, uterus and lung (black). RFX TSS locations without color code have low expression values (TPM < 5). This is followed by the expression profiles of 30 RFX TSS locations in human tissues, primary cells and cell lines, whereby for every one of the eight human RFX genes (1–8), summarized TSS profile data are presented vertically (“top-down”), starting with the a tissue plot, followed by a table of the top 10 tissues, a table of the top 10 primary cells and a table of the top 10 cell lines (highest expression levels are listed first, respectively). The tissue plot is the expression level in log (base 10) TPM against tissues that are sorted from the highest to the lowest expressed from 135 tissues, whereby the plot only includes the first 100 tissues. The arbitrary unit for detection of expression is tags per million (TPM) as defined by FANTOM5. We consider TPM < 5 to be lowly expressed and TPM < 1 to be background noise. (PDF 3276 kb

A–D. Oxidation of NADPH by the thioredoxin and glutaredoxin systems in the presence of selenite and GS-Se-SG, catalyzed by SAM.

<p>Reaction catalyzed by the thioredoxin system was performed in 50 mM Tris-HCl, 1 mM EDTA, pH 7.5, and 200 µM NADPH. A) The reaction mixture contained 2 µM human Trx1 and 50 nM mammalian TrxR1. SAM, 4 mM (♦) Selenite (5 µM) (○), SAM and selenite, 4 mM and 5 µM respectively (without Trx1 in the reaction) (•), SAM and selenite (Δ). B) Reaction catalyzed by the glutaredoxin system was performed in TE-buffer, containing 200 µM NADPH, 6 µg/mL GR, 1 µM human Grx1 and 50 µM GSH. SAM, 4 mM (♦) Selenite (5 µM) (○), SAM and selenite, 4 mM and 5 µM respectively (without Grx1 in the reaction) (•), SAM and selenite, 4 mM and 5 µM respectively (Δ). C) The thioredoxin system with: 5 µM GS-Se-SG (without Trx1 in the reaction) (♦), 5 µM GS-Se-SG (•), 5 µM GS-Se-SG and SAM (without Trx1 in the reaction) (○), 5 µM GS-Se-SG and SAM (Δ). D) The glutaredoxin system with: 15 µM GS-Se-SG (♦), GS-Se-SG and SAM (•), GS-Se-SG and 1 µM Grx1 (○), GS-Se-SG, SAM and 1 µM Grx1 (Δ).</p

A–H. Cytotoxicity of selenium compounds in the presence of SAM.

<p>Cell viability was measured by XTT after 24 h incubation with selenium treatment, combined with SAM, DTNB and MSG. Cells were pretreated with MSG (60 mM) followed by treatment with selenium compounds +/− SAM (500 µM) <b>A</b>) Selenite (5 µM), <b>B</b>) GS-Se-SG (5 µM), <b>C</b>) Seleno-DL-cystine (100 µM). <b>D</b>) SAM toxicity was determined by clonogenic assay. Cells were treated for 8 h with 500 µM SAM, washed and re-seeded, in triplicates. After 9 days, clones were stained and counted. <b>E</b>) Viability over time (0–48 h) after pretreatment with MSG followed by addition of selenite +/− SAM. <b>F</b>) Selenium accumulation in ng/mg protein after 24 h treatment (same concentration of all compounds as in <b>E</b>) measured by GF-AAS analysis. <b>G</b>) Comparison of toxicity between selenite (5 µM) +/− SAM and MSA (5 µM) after 24 h of treatment. <b>H</b>) Representative morphological changes associated with the treatments of selenite (5 µM), selenite +/− SAM and MSA (5 µM) for 20 h. In <b>D</b>, Student t-test was performed to verify the statistical significance between two groups. One-way ANOVA (99.9% confidence interval) followed by Tukey-Kramer multiple comparison test was performed to determine statistical significance in <b>A–C, F</b> (**p<0.01 and ***p<0.001 compared to controls, °p<0.01 and °°p<0.001 compared to selenium treated cells). In <b>E</b>, two-way ANOVA (95% confidence interval) was performed, followed by Bonferroni multiple comparison test. (*p<0.05 and ***p<0.001, compared to control at selected time point). In Fig. <b>G</b>, one-way ANOVA was used, followed by Student-Newman-Keuls multiple comparison test (95% confidence interval, *p<0.05 compared to selenite treatment).</p