Data_Sheet_1_Dynamic changes in the home range of the subterranean rodent Myospalax baileyi.zip

As ecosystem engineers, subterranean rodents excavate and inhabit burrow systems. However, the changes in their use of underground space are poorly recorded. There is conflicting evidence about whether the burrow systems of subterranean rodents, once established, are relatively stable as a result of the high energy costs of digging. We monitored the size of the home ranges of the plateau zokor (Myospalax baileyi) during different stages of its life cycle to show whether mating behavior and the characteristics of its habitat influence the size and location of its home range. We used radio-tracking to quantify the changes in, and overlap of, the home range of M. baileyi during a one-year period. The average size of the home ranges of male zokors was 6.5 times larger than that of female zokors during the mating season. The males expanded their burrows to overlap with multiple females to increase their chances of mating. However, there was no overlap between estrus females or males, perhaps to reduce the number of encounters and unnecessary fights. The home ranges of male and female zokors were similar in size after courtship and the home ranges of single zokors overlapped with those of several neighbors. Most individuals remained territorial and excluded intraspecific interactions from their home ranges. The location of female zokors was stable throughout the year, but half of the males changed the location of their nests and established completely new home ranges in the non-breeding season, mainly in October. The use of space by M. baileyi is flexible in response to a need for physical contact during the mating season and food resources. The home ranges of subterranean plateau zokors are dynamic and the home ranges of male zokors can change within one breeding cycle.</p

Comparison of gene expression profiles of CD4+ T cells transduced with Foxp3 K17R versus K18R.

<p>Heat maps showing distinct gene expression profiles of CD4+ T cells transduced with WT Foxp3, K17R, K18R or EV.</p

Single Foxp3 lysine mutations affect Treg suppressive function and gene expression.

<p>(A) CD4+ CD25− T cells transduced with retroviruses encoding WT Foxp3, K16R, K17R, K18R, K19R or EV; Foxp3 staining showed >80% transduction efficiency. (B) Effects of single lysine mutations on Treg suppressive activity. (C) RNA derived from CD4+CD25− T cells transduced with WT Foxp3, K17R, K18R or EV were analyzed for CTLA4 (in the absence of CD3/CD28 mAbs) and IL-2, IL-4, IL-17 and IL-21 (in the presence of CD3/CD28 mAbs) gene expression by qPCR. Data were normalized to 18S; *p<0.05, **p<0.01 compared to WT Foxp3. Graphs show means ± SD and results are representative of 3 independent experiments.</p

Helios expression by human Tregs did not correlate with CD31 or suppressive function.

<p>(A) Two liver transplant patients underwent serial analysis of Treg suppressive function, Foxp3 methylation and cell phenotype. At 3 mths post-transplant, Tregs had increased thymic output of CD31+ Tregs and reduction of Helios+ Tregs; Helios was not co-expressed with CD31. (B) Tregs, CD4+CD25- Teffs and CD4-depleted APC from healthy donor were used in suppression assays, with or without treatment with a DNMTi (Decitabine) or HDACi (SAHA). Enhancement of Treg suppression by either agent (left) was not accompanied by change in Helios expression (right).</p

Foxp3 lysine mutations affect Treg suppressive function and gene expression.

<p>(A) Comparison of amino acid sequences of the C-terminal regions of mouse (m) and human (h) Foxp3, with FKH domains in gray. Lysine at position 332 is K16 and the FKH domain contains K17–K21 (consecutive lysines numbered in green), with residues important for Runx1 (R), DNA (D) or NFAT (N) binding indicated in purple and non-conserved residues shown in red; adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029035#pone.0029035-Tao1" target="_blank">[16]</a>. (B) CD4⁺CD25⁻ T cells were transduced with retroviruses encoding WT Foxp3, Foxp16-19R, Foxp16-19Q or EV; Foxp3 staining showed >85% transduction efficiency (%transduced cells shown in blue in each panel). (C) In vitro Treg suppression assays in which 5×10⁵ CFSE-labeled Teff cells were stimulated for 72 h with CD3 mAb in the presence of 5×10⁵ irradiated APC and the indicated ratios of Treg to Teff cells. Data are mean ± SD of duplicate measurements of the percentages of dividing Teff cells, and results are representative of 3 independent experiments; *p value<0.05, **p<0.01 compared to empty vector (EV) in left panel or compared to WT Foxp3 in right panel. (D) RNA derived from CD4⁺CD25⁻ T cells transduced with WT Foxp3, Foxp3 K16-19R, K16-19Q or EV were analyzed for CTLA4, GITR, IL-2, and IL-10 gene expression by qPCR and data were normalized to 18S; *p value<0.05, **p<0.01 compared to WT Foxp3. Graphs show means ± SD and results are representative of 3 independent experiments.</p

Foxp3 mutants impair Foxp3 DNA binding ability.

<p>293T cells were transfected with EV, WT Foxp3, K17R or K18R without or with p300 expression vectors, and 48 h later, cell lysates were harvested. (<b>A</b>) Equal amounts of cell lysates were incubated with biotin-labeled Foxp3 binding site nucleotide, and Foxp3 DNA binding was detected with anti-Foxp3 or anti-acetyl-lysine Abs. The protein expression levels of Foxp3 and loading control β-actin were detected by western blotting; arrow indicates acetylated Foxp3 bound to DNA, and star indicates non-specific binding. (<b>B–D</b>) The densities of Foxp3 DNA-binding bands were measured using Image-J software and normalized with Foxp3 input levels. (<b>B</b>) The relative Foxp3 DNA binding ability in the absence of p300 is shown. (<b>C</b>) Foxp3 and mutant DNA binding ability was increased in the presence of p300. (<b>D</b>) Comparison of relative Foxp3 DNA binding between WT and mutants in the presence of p300 is shown. (<b>E</b>) Comparison of relative acetylated Foxp3 binding level between WT and mutants is shown. Results are representative of 2 independent experiments.</p

Foxp3 mutants impair Treg function in vivo.

<p>(<b>A</b>) 1×10⁶ Thy1.1+ CD4+CD25− T cells were co-transferred with 1×10⁶ Thy1.2+ CD4+ T cells transduced with WT Foxp3, K16-19R, K17R, K18R or EV, or with purified normal B6 Treg cells, into Rag1−/− mice. At 7 d post-transfer, single-cell suspensions from lymph node or spleen samples were stained for FACS analyses; the numbers (<b>A</b>) or percentages (<b>B</b>) of CD4+ Thy1.1+ cells are shown. Results are representative of 2 independent experiments, and *p<0.05 compared to WT Foxp3.</p

Comparison of gene expression profiles of CD4+ T cells transduced with WT Foxp3, K17R, K18R or EV.

<p>(<b>A</b>) Venn diagrams summarizing overlapping upregulated (left) or downregulated (right) gene expression profiles of CD4+ T cells transduced with WT Foxp3 (blue), K17R (purple) or K18R (yellow) compared with CD4+ T cells transduced with EV (cutoff was set as Log2 fold >1). (<b>B–E</b>) Heat maps showing gene expression profiles of CD4+ T cells transduced with EV, WT Foxp3, K17R or K18R. (<b>B</b>) Heat maps showing distinct Treg ‘signature’ gene expression profiles. (<b>C</b>) Heat maps indicating the change in gene expression profiles of Treg cell identified and putative suppressive genes. (<b>D</b>) Heat maps indicating the distinct gene expression profiles of Foxp3 directly bound genes. (<b>E</b>) Heat maps indicating distinct gene expression profiles of Treg-related cytokine genes. (<b>F</b>) qPCR assays of Treg-specific genes selected from microarray analyses; results are representative of 3 independent experiments, and *p<0.05, **p<0.01 compared to WT Foxp3.</p

Helios expression in Tregs in different ages.

<p>Helios expression in Tregs in different ages.</p

Helios is associated with an activated non-mature phenotype in suppression assays.

<p>After suppression assays (1/2 Treg/responder ratio), human CD4+ Teffs (A) and Tregs (B) were divided into 4 subsets according to Helios and Foxp3 co-expression. Helios+Foxp3- cells had the least mature and the most activated phenotypes in human CD4+ Teffs (A) and Tregs (B) in suppression assays, while Foxp3 expressing cells were enriched for mature CD45RO+ CD45RA- subsets. Data are representative of 2 experiments.</p