qRT-PCR primer sequences used in the biotinylated oligonucleotide pull down assay.

<p>qRT-PCR primer sequences used in the biotinylated oligonucleotide pull down assay.</p

Generation of a Sox4 conditional activation system.

<p>The hormone binding domain of the ER was fused to the N-terminus of Sox4. (<b>A</b>) ER:<i>Sox4</i> was stably transduced in U2OS cells. Immuno-fluorescence analysis of ER:Sox4 localization using an anti-ER antibody after stimulation with 4-OHT (100 nM) for the time points indicated. (<b>B</b>) U2OS cells expressing ER and ER:Sox4 were transfected with an optimal Sox4 luciferase reporter construct and treated overnight with 4-OHT (100 nM) after which luciferase activity was measured. (<b>C</b>) ER:Sox4 localization in HMLE cells in presence and absence of 4-OHT. (<b>D</b>) HMLE cells expressing ER and ER:Sox4 were transfected with an optimal Sox4 luciferase reporter construct and stimulated overnight with 4-OHT (100 nM) after which luciferase activity was measured. Confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p

SOX4 expression is increased by TGF-β during EMT.

<p>(<b>A</b>) MARA analysis predicts Sox activity during EMT in HMEC and NMuMG cells (see text for details) (<b>B</b>) Public microarrays databases generated in non-transformed HMEC cells treated with TGF-β or left untreated were analyzed and SOX4 expression was assessed. (<b>C</b>) HMLE cells were stimulated with 2.5 ng/mL of TGF-β as indicated, lysed and mRNA expression of <i>CDH2</i> (N-cadherin), <i>VIM</i> (vimentin), <i>CDH1</i> (E-cadherin) were analyzed by qRT-PCR. (<b>D</b>) HMLE cells were stimulated with 2.5 ng/mL of TGF-β as indicated and lysed. The protein lysates were visualized by Western-Blotting using anti-N-cadherin, anti-SOX4, anti-Tubulin and anti-E-cadherin antibodies. Western blot data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p

Sox4 activation induces upregulation of mesenchymal markers.

<p>(<b>A</b>) HMLE cell lines expressing ER:Sox4 were stimulated with 4-OHT (100 mM) as indicated. Cells were lysed and mRNA expression of <i>CDH2</i> (N-cadherin), <i>VIM</i> (vimentin), <i>FN1</i> (fibronectin) and <i>CDH1</i> (E-cadherin) was analyzed by qRT-PCR. (<b>B</b>) HEK293T cells were transiently transfected with Flag-tagged Sox4 Wt or Flag-tagged Sox4 1-135aa and co-transfected with a <i>CDH2</i> luciferase reporter construct as indicated. After 48 hours luciferase activity was measured. Protein expression was assayed by Western blotting using anti-Flag antibody. (<b>C</b>) Schematic representation of the <i>CDH2</i> promoter region and predicted Sox4 binding sites. (<b>D</b>) Chromatin Immunoprecipitation (ChIP) assay using IgG and SOX4 antibodies in MDA-MB-231 cells. Real time PCR was performed using <i>CDH2</i> promoter-specific primers to test SOX4 occupancy at this region. (<b>E</b>) HEK293T cells were transiently transfected with the empty vector pcDNA3 or Flag-tagged Sox4 Wt. After 48 hours cells were harvested and nuclear fraction was extracted. Nuclear extracts were used to perform a biotinylated oligonucleotide pull down assay in which three <i>CDH2</i> promoter sites and two sites localized in the first intron of <i>CDH2</i> were included. Lysates were assessed by western blotting using anti-Flag antibody. (<b>F</b>) HMLE cell lines expressing ER:Sox4 were stimulated with 4-OHT (100 nM) as indicated or left untreated. Cells were lysed and lysates were analyzed by Western blotting using anti-N-cadherin, anti-Tubulin, anti-E-cadherin and anti-ER antibodies. (<b>G</b>) HMLE cells expressing ER:Sox4 were treated with 4-OHT (100 nM) as indicated. Cells were fixed, permeabilized and the expression of N-cadherin and E-cadherin was assessed (green and red respectively). Blue = DAPI. Western blot and confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p

SOX4 knockdown delays TGF-β-induced EMT.

<p>HMLE cells line were either transduced with a control shRNA (Scr shRNA) or with a shRNA targeting SOX4 (SOX4 shRNA). (<b>A</b>) Scr shRNA and SOX4 shRNA cell lines were either treated with 2.5 ng/mL of TGF-β for 7 days or left untreated. Cells were lysed and analyzed by Western Blotting using anti-SOX4 and anti-Tubulin antibodies (<b>B</b>) HMLE cell lines expressing Scr shRNA and SOX4 shRNA were stimulated with 2.5 ng/mL of TGF-β as indicated. Cells were lysed and mRNA expression of <i>CDH2</i> (N-cadherin), <i>VIM</i> (vimentin) and <i>CDH1</i> (E-cadherin) were assessed. (<b>C</b>) Cell lysates of HMLE cell lines expressing Scr shRNA and SOX4 shRNA stimulated with 2.5 ng/mL of TGF-β as indicated and analyzed by western blotting using N-cadherin, anti-Tubulin and anti-E-cadherin antibodies. (<b>D</b>) Scr shRNA and SOX4 shRNA cell lines were stimulated with 2.5 ng/mL of TGF-β as indicated. Cells were fixed, permeabilized and N-cadherin expression was assessed (green). Blue = DAPI; red = phallodin. Western blot and confocal microscopy data is representative of at least three independent experiments. *p<0,05 (N = 3±SD).</p

SIRT1 inhibition increases Foxp3 transcriptional output.

<p>(<b>A</b>) Utilizing an IL-2 promoter reporter, Foxp3 transcriptional function was analyzed. HEK293 cells were transfected with an IL-2 promoter luciferase reporter, NFAT, Foxp3, Foxp3 K22xR and SIRT1, cells were lysed and assayed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019047#s2" target="_blank">Materials and Methods</a>. Luciferase values were normalized for co-transfected renilla to correct for transfection efficiency. (<b>B</b>) iTregs were generated by culturing human CD4+CD25- human T cells with anti-CD3 and anti-CD28 coupled beads, in combination with 10 ng/ml TGF-β and 300 U/ml IL-2 for 5 days. At day 0, 3, 5 cells were harvested and mRNA expression of SIRT1 and Foxp3 was analyzed by qRT-PCR. (<b>C</b>) Freshly isolated human CD4+ T cells were cultured in the presence of 300 U/ml IL-2, anti-CD3 and anti-CD28 and 0, 1 or 9 mM NAM for three days. mRNA expression of multiple transcriptional targets of Foxp3 was analyzed by qRT-PCR. Data are representative of at least three independent experiments, * P<0.05, ** P<0.01.</p

Q-PCR primers.

<p>Q-PCR primers.</p

Nuclear association of Foxp3 and SIRT1.

<p>(<b>A</b>) HEK293 cells were co-transfected with both HA-Foxp3 and Flag-SIRT1, lysed, and Foxp3 was immunoprecipitated and association of proteins was analyzed by Western blotting utilizing anti-Flag antibodies. (<b>B</b>) HA-Foxp3 and Flag-SIRT1 transfected cells were lysed, SIRT1 was immunoprecipitated and the association of Foxp3 was assessed as in (<b>A</b>). (<b>C</b>) SIRT1-Foxp3 interaction in transfected HEK293 cells was visualized using <i>in situ</i> proximity ligation assay (PLA). Cells were fixed and protein-protein interactions were visualized utilizing anti-HA and anti-Flag antibodies as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019047#s2" target="_blank">Materials and Methods</a> section. Punctate staining (red) indicates Foxp3-SIRT1 interaction as detected by the assay. Nuclei were stained using Hoechst. Representative images from at least three independent experiments are depicted.</p

SIRT1 regulates Foxp3 degradation.

<p>(<b>A</b>) HEK293 cells were co-transfected with HA-Foxp3 and Flag-SIRT1 and treated with 2 µM MG132 for 16 hours. Cells were lysed and Foxp3 protein levels were visualized by Western blotting using anti-HA antibodies. (<b>B</b>) Cells were transfected with HA-Foxp3 and treated with 20 mM NAM for 16 hours, Foxp3 protein levels were analyzed by Western blotting using anti-HA antibodies. (<b>C</b>) Cells ectopically expressing HA-Foxp3 were treated with both 20 mM NAM and 5 µg/ml cyclohexamide (CHX) for 16 hrs and Foxp3 levels were analyzed by Western botting. (<b>D</b>) HA-Foxp3 or a HA-tagged Foxp3 mutant in which all lysines were mutated to argenines (HA-Foxp3 K22xR) was transfected into HEK293 cells. Cells were treated with 20 mM NAM for 16 hours. Cell lysates were prepared and Foxp3 levels were evaluated by immunoblotting for HA and HSP90 as control. (<b>E</b>) CD4+ T cells isolated from human PBMC were cultured in the presence of 300 IU/ml IL-2, 2.0 µg/ml anti-CD28, and 1.5 µg/ml plate-bound anti-CD3 and treated NAM (1 or 9 mM). After 4 days, the percentage of Foxp3+CD25+ cells was analyzed by FACS. (<b>F</b>) Histogram of the data shown in (<b>E</b>). Data are representative of at least three independent experiments.</p

SIRT1-mediated deacetylation increases Foxp3 poly-ubiquitination.

<p>(<b>A</b>) HEK293 cells ectopically expressing HA-Foxp3 were transfected with Flag-SIRT1. Cell lysates were prepared and HA-Foxp3 was immunoprecipitated. Acetylated Foxp3 was visualized by Western blotting using anti-acetylated lysine antibodies. (<b>B</b>) Analysis of Foxp3 acetylation after treatment with 20 mM nicotinamide (NAM) for 16 hours as in (<b>A</b>). (<b>C</b>) Cells were transfected with Flag-Foxp3 and treated with 20 mM NAM (for three hours), Foxp3 was immunoprecipitated from cell lysates and ubiquitination was analyzed by Western blotting utilizing anti-ubiquitin antibodies. * Indicates aspecific band. Data shown are representative of three independent experiments.</p