B cells and macrophages fail to induce transcriptional enhancer activity of the TSDR.

<p>Dual luciferase assays were performed after transfecting reporter plasmids carrying the indicated inserts or an empty pGL3 vector (EV) into RLM-11 cells (T cell line), A20 cells (B cell line) or RAW 264.7 cells (macrophage cell line). Three hrs (RLM-11, A20) or 20 hrs (RAW 264.7) after transfection, cells were stimulated for 16 hrs with PMA/iono (RLM-11, A20) or for 14 hrs with LPS/IFN-γ (RAW 264.7), followed by measurement of luciferase activities (mean±SD, n = 3). Data are representative of two to four independent experiments.</p

The postulated NF-κB binding site of the TSDR is not transcriptionally responsive to activated NF-κB.

<p>(<b>A</b>) RLM-11 cells were stimulated with PMA/iono for indicated time periods and applied to subcellular fractionation. Nuclear and cytoplasmic extracts were analyzed by Western blotting using the indicated antibodies. p44/42 and lamin B served as loading and purity controls for cytoplasmic and nuclear fractions, respectively. (<b>B</b>) A luciferase reporter plasmid integrating the NF-κB-RE was transfected into RLM-11 cells and dual luciferase assays were performed in triplicates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a>. Mean luciferase activity is shown as fold increase to unstimulated control. Results are representative of four independent experiments. (<b>C</b>) A schematic view on the first part of the <i>Foxp3</i> gene locus is depicted. White boxes indicate untranslated exons, the first translated exon is indicated in black. Evolutionary conserved non-coding sequences (CNS) are indicated in grey. The distended region of the TSDR includes the previously described NF-κB binding site (black frame), which is flanked by the seventh CpG motif (underlined) of the TSDR. (<b>D</b>) A tandem of five repetitive sequences of the putative NF-κB binding site was inserted into the pCpGL luciferase reporter plasmid upstream of the <i>EF</i> promoter (tandem-EFPro). Dual luciferase assays were performed as described in (B) using pCpGL-TSDR-EFPro as a positive control. Data represent one out of two independent experiments.</p

Degradation of IκBα is not required for TSDR enhancer activity.

<p>Luciferase plasmids integrating either the NF-κB-RE or TSDR-FoxPro were co-transfected with either an empty vector or with a vector encoding the super-repressor, a non-degradable form of IκBα, into RLM-11 cells. Dual luciferase assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088318#pone-0088318-g001" target="_blank">Figure 1</a> and unstimulated cells served as controls. Luciferase activities are shown as percent of empty vector controls and standard deviations of performed triplicates are shown. One representative experiment out of at least two independent experiments is depicted.</p

c-Rel^−/− Tregs show a stable phenotype.

<p>(<b>A</b>) CD4⁺CD25^hi Tregs and CD4⁺CD25⁻ Tconv were isolated from wild-type (WT) or c-Rel^−/− mice. Genomic DNA was isolated and subjected to bisulfite sequencing in order to determine the methylation status of CpG dinucleotides within the TSDR. (<b>B</b>) CD4⁺CD8⁻CD62L^hiCD25^hi Tregs from spleen and lymph nodes of c-Rel^−/− or WT mice were sorted and an aliquot was analyzed for Foxp3 expression by flow cytometry (top panel). Cells were cultured in the presence of IL-2 and stimulated by plate-bound α-CD3/CD28 for six days followed by flow cytometric analysis of Foxp3 expression. Cells depicted were pregated to viable CD4⁺ T cells. Results represent one out of two independent experiments.</p

Kinase activity of IKKα and IKKβ is largely dispensable for TSDR enhancer activity.

<p>(<b>A</b>) Luciferase plasmids encoding NF-κB-RE or TSDR-FoxPro were co-transfected with plasmids encoding kinase dead (KD) or wild-type (WT) forms of IκB kinase α and β (IKKα and IKKβ) into RLM-11 cells. Cells were cultured for one day allowing efficient protein expression before cells were stimulated overnight with PMA/iono and dual luciferase assays were performed. Luciferase activities are given as percent of luciferase activity of WT samples and standard deviations were calculated from three replicates. (<b>B</b>) Dual luciferase assays as described in (A) were performed co-transfecting the indicated luciferase constructs with a plasmid encoding the constitutively active form of IKKβ (IKK-CA) or empty vector as control (mean±SD, n = 3). One representative out of three independent experiments is shown.</p