Custom Track View of DNase I hypersensitive sites (DHS) sites.

<p>DHS sites, Pol II, H3K20me1, H3K4me3, H3K4me2, H3K4me1, H2BK5me1 and H2A.Z along the FRA2 gene on Chr 2 were obtained from the UCSC Genome Browser.</p

Gene ontologies of putative STAT5 targets from activated CD4+T cells.

<p>Gene function of putative binding sites is based on the summary information provided in the PANTHER database. Out of the 105 binding sites identified, functional categorisation was done for 104 genes, out of which 99 could be classified and the remaining 5 genes were of unknown/uncategorized function. Those 99 genes were classified based on their molecular function (A) and biological processes (B). In some instances, a given gene was represented in more than one category (for example, FRA2 was incorporated under the binding (GO:0005488) as well as transcriptional regulator activity (GO: 0030528) molecular functions).</p

IL-2 regulates expression of FRA2.

<p>Quantitative RT-PCR was performed on PHA activated CD4+ T cells stimulated with or without IL-2 for different times to evaluate the expression of FRA2. Expression levels are presented as fold increase (logarithmic scale) and compared to the baseline levels (cells not treated with IL-2). 18 s was used as the housekeeping gene and served as the endogenous control. IL-2 stimulation strongly induced <i>FRA2</i> expression with two peaks observed at 4–6 hours and 24 hours post stimulation. This is representative of at least three independent experiments performed in triplicate.</p

TCR induced FRA2 expression is dependent on IL-2 signaling.

<p>CD4+ T cells were TCR activated ±HAT (humanized anti-Tac antibody) treatment and mRNA was prepared pre- and 17 hours post-stimulation. qRT-PCR analysis for FRA2 expression was carried out. TCR activation induces FRA2 expression, which is abrogated by the addition of HAT, indicating that IL-2R function is essential for induction of FRA2. This is representative of at least three independent experiments performed in triplicate.</p

STAT5 motif analysis of the mapped ChIP clones in activated CD4+ T cells.

<p>Out of the 105 binding sites for activated CD4+ T cells immunoprecipitated with anti-STAT5 Ab, 68% had TTN₅AA motifs and TTCN₃GAA sites were present in 26% of the target sites. The labels denote the category name and percentage.</p

Activation of the JAK3-STAT5 pathway is essential for the induction of FRA2 gene expression by TCR activation.

<p>CD4+ T cells were stimulated via the TCR ± the JAK3 inhibitor R333, RNA was prepared four hours post treatment. qRT-PCR analysis was performed to detect the relative expression of FRA2. FRA2 gene expression was induced by TCR activation and abrogated by R333 treatment. Shown is a representative experiment performed in triplicate and repeated three times.</p

Transcriptional activities of the GAS1 sequence.

<p>Trimerised GAS1–luciferase reporter was cotransfected with IL-2R components in 293T cells, as described in materials and methods. The results are from three independent experiments performed in triplicate. This shows that the GAS1 sequence identified by the ChIP studies can be specifically activated by STAT5 in an IL-2-receptor reconstitution assay in 293T cells.</p

Study of the binding site, location and validation of the cloned fragment obtained by ChIP.

<p>A. Sequence of the cloned fragment and the corresponding typical and atypical GAS sequences present. There is a TTN₅AA (blue font) sequence and one typical GAS TTCN₃GAA (red font) sequence present within the cloned fragment. B. The relative location of the cloned fragment, GAS1 and GAS2 with respect to the FRA2 transcription start site (TSS) and intron/exon is shown schematically and its coordinates on the UCSC genome browser. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090370#pone-0090370-g004" target="_blank">Figure 4C:</a> IL-2 induces in-vivo binding of STAT5 to GAS1 within the third intron of the FRA2 gene in preactivated CD4 T cells. STAT5-ChIP samples from ±IL-2-treated preactivated CD4 T cells were analysed by real-time PCR for specific binding to the two GAS motifs identified above. ChIP assay was performed using the LowCell# ChIP kit and ±IL-2-treated preactivated CD4 T cells. Enrichment of GAS 1 was higher when compared to GAS 2 and more so with the STAT5b antibody.</p

The locations of putative STAT5 binding sites in activated CD4+ T cells.

<p>Mapping of STAT5 binding sites relative to annotated genes were categorized into four groups. Intergenic region denotes binding sites present greater than 10<10 kb upsteam denotes the region less than 10 kb upstream of the 5′ region of the nearest gene. Internal intron denotes introns other than intron 1 of the gene and intron 1 depicts the presence of a binding site within the first intron of the gene.</p

<i>C. albicans</i> infection of A431 epithelial cells activates NF-κB and MAPK signaling.

<p><i>C. albicans</i> SC5314 was added to A431 vaginal ECs under standard culture conditions for 5, 15, 30, 60 min and 2 h. Total protein was isolated and phosphorylation of (A) IκBα or (B) p38, JNK, ERK1/2 and MKP1 were assessed by Western blotting. Bands are shown with an α-actin loading control. A <i>C. albicans</i>:EC MOI of 10∶1 was used. Data are representative of three independent experiments.</p