The human RSUME splice variants.

<p>Schematic illustration of the RSUME pre-mRNA (above) and the four human RSUME mRNA transcripts splice variants documented to the date. The variant 1 (NM_015485) encodes the longest isoform of RSUME containing 267 aminoacids (RSUME267); the variant 2 (NM_ 001128142) encodes the shortest isoform of RSUME containing 195 aminoacids (RSUME195); the variant 3 (NM-001199682) encodes a 200 aminoacids RSUME (RSUME200); and the variant 4 (NR_037643) is a non-coding RNA because it presents a premature stop codon in the exon 2 that goes to degradation via NMD. Boxes, exons with the exon number inside; filled black lines, introns; gray lines, splicing events (filled, splice event that originates the variant 1; dashed, variant 2; dotted, variant 3; streak and point, variant 4); +1, transcription start site; AUG, translation start site; UAA, UGA and UAG, translation stop sites. This information was obtained from published sequences in the University of California, Santa Cruz, genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>).</p

Effect of RSUME195 and RSUME267 over HIF-1 signaling pathway.

<p><b>A.</b> COS-7 cells were co-transfected with 500 ng of HRE-LUC report vector, 300 ng of Gaussia report vector and different concentrations of RSUME195 or 267 (100 or 300 ng) to evaluate their effect in HIF-1 transcriptional activity. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). ^#, p<0.05 compared each concentration of cells, under hypoxia, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. <b>B.</b> COS-7 cells were co-transfected with 300 ng of Flag-HIF-1alpha and/or 500 ng of V5-RSUME195 or V5-RSUME267, or the corresponding empty vector (were both are absent). Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. Cell extracts were subjected to western blot. NMX, normoxia; HPX, hypoxia; Vect, cells transfected with the corresponding empty vectors. <b>C.</b> COS-7 cells were co-transfected with RSUME and HIF-1alpha. Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. RIPA cell extracts were inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. <b>D.</b> COS-7 cells were co-transfected with 500 ng of VEGF-LUC report vector, 300 ng of CMV-β Gal report vector and RSUME195 or 267. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to β-galactosidase value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) under hypoxia (ANOVA with Scheffè’s test). ^#, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267, under hypoxia (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. <b>E.</b> Semi-quantitative RT-PCR of endogenous VEGF and β-actin mRNA in HepG2 cells transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O_2, 5% CO₂ and 94% N₂) for 16 h, twenty-four hours after transfection. <b>F.</b> VEGF mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HeLa cell stimulated with hypoxia for 16 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). ^#, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). <b>G.</b> HepG2 cells were transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O_2, 5% CO₂ and 94% N₂) for 16 h, twenty-four hours after transfection. VEGF protein levels were analysed by western blot.</p

RSUME267 and RSUME195 similar actions on the SUMO pathway.

<p><b>A.</b> 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from <i>E.coli</i> transformed with the empty pQE30 vector. <b>B.</b> 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. <b>C.</b> Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.</p

mRNA and protein expression of RSUME195 and RSUME267 in cells and tumors.

<p><b>A.</b> Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 (left panel) and RSUME267 (right panel), was performed in COS-7 cells exposed to normoxic or hypoxic conditions (1% O_2, 5% CO₂ and 94% N₂). <b>B.</b> Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267, was performed on an unstimulated human non-functioning pituitary tumor explant sample. <b>C.</b> Semi-quantitative RT-PCR with specific primers to detect mRNA levels of RSUME195 and RSUME267 was performed on human glioma samples. <b>D.</b> Western blot analysis of RSUME protein levels was performed on human glioma samples. β-actin protein levels were used as control.</p

Domain characterization of RSUME.

<p><b>A.</b> Secondary structure prediction with PHYRE Server of the whole RSUME267 amino acid sequence. <b>B.</b> Ribbon representation of the RSUME267 structure as predicted by the PHYRE Server, showing the secondary structure elements: alpha helixes (purple), beta-sheets (yellow), loops (cyan). The Figure was made with the program VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057795#pone.0057795-Humphrey1" target="_blank">[29]</a>. <b>C.</b> The same structure that in <b>B</b> but different colors encompassing the different regions of the RSUME267 protein. Blue residues 1 to 123, red residues 124 to 137, green residues 137 to 195, brown residues 196 to 267. <b>D.</b> Schematic comparison of the domains present in the three RSUME isoforms and in the RWD domain containing superfamily. Boxes, structural domains; lines, loops or unstructured regions; numbers, amino acid position.</p

Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.

<p><b>A.</b> COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. <b>*</b>, band corresponding to an IgG used in the inmunoprecipitation. <b>B.</b> and <b>C.</b> COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (<b>B</b>), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (<b>C</b>), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). ^#, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). <b>D.</b> IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). ^#, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).</p

RSUME structural features.

<p><b>A.</b> BLAST multiple sequence alignment for RSUME267 (NP_056300), RSUME195 (NP_001121614) and RSUME200 (NP_001186611) isoforms. The identical aminoacids in the alignment are showed in gray. <b>B.</b> Ribbon Representation showing the secondary structure elements (alpha helixes are purple, beta-sheets are yellow and loops are cyan) of the RWD domain of RSUME obtained from the PDB (PDBid: 2EBK). The figure was made with the program VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057795#pone.0057795-Humphrey1" target="_blank">[29]</a>.</p

EDA⁺ but not EDA^- peptide increases the proliferation rate of mESC.

<p>(A) Ainv 15 mouse ES cells were cultured in the standard media supplemented by the corresponding peptide preparation to a final dose of 10 µg of protein per ml of medium for 72 hours. (EDA-containing recombinant peptide, EDA⁺; EDA-lacking recombinant peptide, EDA^-). Representative brightfield pictures are shown. Bars correspond to 50 µm. (B) The proliferation of Ainv15 mES cells cultured in the media described in B for 48 or 72 hours, as indicated, was evaluated by crystal violet. A representative experiment of at least three replicates is shown. Bars represent mean ± SD. Statistical analysis was done by One way ANOVA with Bonferroni test for multiple comparisons. * indicate significant differences between treatments (<i>P</i> < 0.05); ** indicate significant differences between treatments (<i>P</i> < 0.005).</p

MEF conditioned medium that contains FN EDA⁺ increases the proliferation rate of mESC.

<p>Ainv15 mES cells were cultured in the media conditioned by wild type MEF (wt), EDA^+/+ MEF (^+/+) or EDA^-/- MEF (^-/-) for 2 days. The proliferation was evaluated by MTT assay. A representative experiment of at least three replicates is shown. Bars represent mean ± SD. Statistical analysis was done by an unpaired t-test. * indicate significant differences between treatments (<i>P</i> < 0.01).</p

EDA –treated ES cells express pluripotency markers.

<p>WA09 human ES cells and Ainv15 mouse ES cells were cultured for three days on feeder free conditions in the indicated medium (untreated cells, C; EDA^+/+ MEF-CM, CM; EDA+ including peptide, PEP). RNA was extracted and the expression of Oct4, Sox2 and Nanog pluripotency gene markers was analyzed by RT-PCR. The expression of the housekeeping GAPDH gene was used as control.</p