Effects of knockdown of RNASEL, IRF7 and IRF3 on the effects of inducible expression of either mutant (mt) or wild type (wt) mouse <i>Oas2</i> in T47D cells.

<p><b>(A-G)</b> Provide the context of RNase L knockdown. <b>(A-C</b>) Demonstration of Doxycycline (DOX)-inducible expression of wt or mt <i>Oas2</i> in T47D cells, and effective knock-down of RNASEL (RNaL) in mt or wt expressing T47D cells by quantitative PCR (<b>B</b>) or western blot (<b>C</b>). <b>(D)</b> Effect of the induction of mt or wt OAS2 on RNase L activity <b>(E)</b> Effects of induction of mt and wt <i>Oas2</i> expression on apoptosis. <b>(F)</b> Effects of these treatments on interferon gamma protein production. <b>(G</b>) effects of these treatments on GM-CSF production. <b>(H</b>) Demonstration of effective knockdown of IRF7. <b>(I)</b> Effects of knockdown of IRF7 on mutant or wild type <i>Oas2</i>-driven apoptosis. <b>(J)</b> Demonstration of knockdown of IRF3. <b>(K)</b> Effects of knockdown of IRF3 on mutant or wild type <i>Oas2</i>-driven apoptosis.</p

Discovery of a pedigree with dominant inheritance of failed lactation.

<p><b>(A)</b> Lactation performance of dams of the indicated genotypes (wild type; wt/wt mutant; mt/mt) assessed by pup weight-gain or survival (inset). Error bars show standard error of the mean for 4–5 litters per genotype of 7 pups each. wt/wt n = 35, wt/mt n = 28 and mt/mt n = 28 pups. <b>(B and C)</b> Whole mount histology of the 4^th inguinal mammary gland showing lobuloalveolar development at 2 days post partum (dpp) in wt/wt or mt/mt mice. <b>(D and E)</b> Corresponding haematoxylin-eosin histochemistry. <b>(F and G)</b> Corresponding immunohistochemistry for milk protein expression. <b>(H)</b> Corresponding Western blot for milk proteins. Molecular size is shown together with the established sizes of the indicated milk proteins [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007072#pgen.1007072.ref035" target="_blank">35</a>]. Lactoferrin (LF), serum albumin (SA), caseins α,κ,β,γ and ε, whey acidic protein (wap) and alpha lactalbumin (lac). <b>(I)</b> Quantification of <i>Wap</i> mRNA by qPCR at in wt/wt or mt/mt mice. <b>(J)</b> Quantification of β-casein (β-Cas) mRNA by qPCR. <b>(K)</b> Quantification of epithelial cell death by immunohistochemistry for cleaved caspase 3, results are the number of positively stained epithelial cells as a percentage as a percentage of total number of epithelial cells per field. <b>(L</b>) Quantification of epithelial cell proliferation by incorporated BrdU expressed as a percentage of total number of epithelial cells per field. <b>(M and N</b>) immunohistochemistry for phosphorylated (P) STAT1 at 2 days post partum (dpp) in wt/wt or mt/mt mice. <b>(O)</b> quantification of P-STAT1 in wt/wt or mt/mt mice by immunohistochemistry, results are the number of positively stained epithelial cells as a percentage of total epithelial area. <b>(P)</b> Quantification of P-STAT1 in wt/wt or mt/mt mammary transplants by immunohistochemistry, results are the number of positively stained epithelial cells as a percentage of total epithelial area. <b>(I-J and O)</b> wt/wt n = 4–5 mice, mt/mt n = 3–5 mice per time point <b>(P)</b> wt/wt n = 3–5 mice, mt/mt n = 2–5 per time point. Student’s t-test p values are given, error bars are standard error of the mean.</p

The effects of inducible expression of mutant and wild type <i>Oas2</i> in T47D cells.

<p><b>(A</b>) pHUSH ProEx expression vector used to express either mutant (mt) or wild type (wt) mouse <i>Oas2</i> in T47D cells in response to doxycycline (DOX). <b>(B</b>) relative expression of mt and wt <i>Oas2</i>. <b>(C</b>) Western blot showing induction of mouse OAS2 (m) running just below endogenous human OAS2 protein, with both bands above a non-specific band (nsb). <b>(D</b>) Sensitivity of the cells lines to poly I:C (pl:C) with and without DOX induction of mt and wt <i>Oas2</i>. <b>(E</b>) Effect of mt and wt <i>Oas2</i> on adherent cell number after 72h. (<b>F</b>) Cell detachment (numbers of live cells in supernatant fraction) caused by mt <i>Oas2</i>. <b>(G</b>) Effects of mt or wt <i>Oas2</i> on replating of T47D cells in a 4 hour trypsin only replating assay after 48h of DOX. <b>(H</b>) Expression of β1 integrin (β1), E-cadherin (EC) and β-actin (βa) in response to induction of mt or wt <i>Oas2</i>. <b>(I</b>) apoptotic response to induction of mt or wt <i>Oas2</i>. Data represents the average of 7 independent experiments. <b>(J</b>) cell-cycle-phase distribution at the indicated times following induction of mt or wt <i>Oas2</i>. Data represents the average of 5 independent experiments. <i>*</i>p<0.01. ANOVA 4I and J. <b>(K)</b> <i>Oas2</i> expression in parental (p) normal mouse mammary HC11 cells or in cells constitutively expressing mt or wt <i>Oas2</i>. <b>(L)</b> Effect of wt or mt Oas2 on beta Casein in HC11s after 72 hours of prolactin (Prl) and Dexamethasone (Dex) stimulation. <b>(M)</b> Effect of mt or wt <i>Oas2</i> expression on cell death at 96 hours in HC11 cells after transient transfection. All data are representative of 3 independent experiments in response to 72h of DOX except otherwise specified. Paired t-tests 4B,E,F, G, L and M.</p

Effects of OAS2 mutation on global patterns of gene expression in the mammary gland.

<p>Whole mouse mammary glands from homozygous <i>Oas2</i> mutant (mt) or wild type (wt) animals were profiled using Affymetrix MTA arrays. Differential gene expression was ranked by the limma t-statistic and this was used as the input for gene set enrichment analysis to identify functional signatures. The enrichment-map plug in for Cytoscape was used to visualize the results. Each node represents a gene set and the expression of genes comprising the leading edge of some of these sets is shown as heat maps of the t-statistic. Labels indicate the function of the clustered gene sets. Gene expression in mt animals is compared with wt animals at 2dpp (node center color) or 18dpc (node edge color). Red indicates enrichment of expression the gene set and blue suppression of expression.</p

Taqman probes used for quantitative PCR indicating gene name, probe ID number and species specificity.

<p>Taqman probes used for quantitative PCR indicating gene name, probe ID number and species specificity.</p

Enzymatic properties of mutant OAS2.

<p><b>(A</b>) Details of the mutation in <i>Oas2</i> showing the ENU-induced SNP changing isoleucine to asparagine. <b>(B)</b> RNAseL activity measured as the abundance of RNase L-specific cleavage of tRNA-His-36 (upper panel) or rRNA (lower panel) at day 18 of pregnancy (d18pc) and two days post partum (2dpp). <b>(C</b>) Representative denaturing PAGE separating 2-5A species of different molecular weights synthesized in a cell free system by mutant (mt) or wild type (wt) mouse OAS2, in response to activation by different concentrations of the double-stranded RNA mimic polyI:C. <b>(D</b>) quantification of the data in panel C. <b>(E</b>) western blot demonstrating similar OAS2 protein input to the assay above.</p

ELF5 produces hemorrhagic mammary tumors and increased tumor vasculature.

<p><b>Panel A</b>, appearance of PyMT-driven tumors in WT mice or those experiencing long-term (8 wk) forced expression of Elf5. <b>Panels B–D</b>, increased presence of erythrocytes (H&E), leukocytes (black arrows), and endothelial cells (white arrows), respectively (immunohistochemistry [IHC]), driven by ELF5, (scale bars 100μm). Flow cytometric (FC) quantification of these effects is shown in the right-hand side (RHS) panels. <b>Panel E</b>, measurement by immunofluorescence (IF) of CD31+ endothelium area (blue) in relation to the total cell area stained by DAPI (yellow). ImageJ quantification of random fields is shown in the RHS panel.</p

Elf5 immunohistochemistry as a predictor of luminal breast cancer survival.

<p>ELF5 was measured by immunohistochemistry in the cytoplasm and nucleus of tumors in a subset of ER+ samples from the Nottingham breast cancer series. <b>Panel A</b>, overall survival (OS) and <b>Panel B</b> distant metastasis free survival (DMFS). Hazards ratio (HR) and Log Rank <i>p</i>-value (p) are given for 5, 10, and 15 y of follow-up. Tumors are split into high ELF5 expression (green) and low Elf5 expression (blue) by XTile and <i>p</i>-values are black where >0.1, red where ≤0.05, and pink where 0.05–0.1.</p

ELF5 drives angiogenesis and the generation of an intra-tumor leaky vasculature.

<p><b>Panel A</b>. Intravital real-time microscopy of blood tracer quantum dots (red) injected into the vasculature of mice of the indicated genotypes. LHS panels, quantum dots visualized together with EGFP (green) marking Elf5 expression. RHS panels, imaging of quantum dots (red) in the tumors 30, 60, and 90 min after injection. <b>Panel B</b> quantification of quantum dots in relation to the distance from the center of multiple blood vessels in ELF5 animals (red hues) or control animals (blue hues) at the times indicated. Raw data can be found at <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002330#pbio.1002330.s002" target="_blank">S2 Data</a>. <b>Panel C</b>, statistical analysis of the vascular leakiness revealed by imaging of quantum dots in four and three mice of each genotype, respectively. <b>Panel D</b>, induction of ELF5 protein in a PyMT cell line in response to 48 h DOX treatment transduced with the pHUSH DOX-inducible expression vector (PyMT-pHUSH-Elf5) or a pHUSH empty vector. <b>Panel E</b>, area occupied by a matrigel plug (indicated by dashes) containing long-term DOX exposed PyMT-pHUSH-Elf5 cells. <b>Panel F</b>, endothelial content of the matrigel plugs removed from mice measured by flow cytometry (FC).</p

Immune cell subsets within tumors and the effect of depletion of MDSCs on ELF5-driven lung metastases.

<p><b>Panels A and B</b>, tumor content of myeloid or lymphoid lineage immune cell subsets expressed as either a proportion of total tumor cells (after erythrocyte lysis) or as a proportion of CD45+ hematopoietic cells. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002330#pbio.1002330.s009" target="_blank">S5 Fig</a> shows the gating strategy. <b>Panel C</b>, relative proportions of Ly6G and Ly6C MDSC in mice of the indicated genotypes (Ly6G/Ly6C ratio between genotypes are nonsignificant Student’s <i>t</i> test <i>p</i> = 0,886). <b>Panel D</b>, the effect of ELF5 on the proportion of reactive oxygen species (ROS) positive Ly6C or Ly6G cells (LHS panels) or on ROS cellular intensity measured as geometric mean (RHS panels). Nonsignificant <i>p</i>-values are not shown.</p