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

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

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

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

<i>ELF5</i> expression in normal breast and breast cancer.

<p><i>ELF5</i> expression in the UNC337 breast cancer cohort according to subtype. Top panel shows combined results with statistical analysis (thick black line, median;, box, interquartile range 25%–75% of points; whiskers, 1.5× interquartile range; crosses, individual tumors). Bottom panel <i>ELF5</i> expression in all individual tumor and normal samples. Basal and normal had significantly higher expression of ELF5.</p

ELF5 suppresses the estrogen-sensitive phenotype.

<p>(A) Western blot showing reduced expression of key genes involved in the response to estrogens following induction of ELF5 expression. (B) Reduced transcriptional activity of reporters of <i>ER</i> and <i>FOXA1</i> (<i>UGT2B17</i> promoter) transcriptional activity following induction of ELF5 in MCF7 cells. Black bars, -DOX, grey bars +DOX 72 h for <i>ERE</i> and 24 h and 48 h for <i>FOXA1</i>. (C) Cell accumulation in MCF7-V5 cell cultures with (+E) or without (−E) 10 nM estrogen treatment, or following expression of ER (+ER) and 10 nM E in the context of induced ELF5. Black bars, -DOX; grey bars +DOX, 72 h and 144 h, respectively. (D) interaction of ELF5-regulated gene sets with estrogen-regulated gene sets. <i>p</i>-Values and odds ratios derived from hypergeometric tests. Number of genes in brackets. (E) Enrichment of gene sets in ELF5 ChIP targets either down (Dn) or Up in response to forced ELF5-V5 expression in T47D cells with DOX. P-Values for hypergeometric tests from GSEA (upper case) or Oncomine (lower case).</p

Elf5 modulates the adhesion of breast cancer cells.

<p>(A) Quantification of detached cells in cultures treated with DOX (+D) compared to no induction (−D). (B) Ability of DOX-treated cells to replate 4 h after trypsin destruction of attachment proteins, compared to untreated cells. Data are expressed as a percentage of replated untreated cells. (C) Proportion of apoptotic cells in DOX treated (grey bars) compared to untreated (black bars) T47D-ELF5-V5 cells, measured by flow cytometry using the M30 antibody. (D) Expression and activation of key cell adhesion proteins following DOX induction of ELF5-V5 expression.</p

Visualization of the transcriptional functions of ELF5 in breast cancer.

<p>Affymetrix transcript profiling following induction of ELF5 in T47D and MCF7 luminal breast cancer cells, analysed by LIMMA and GSEA. <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#s2" target="_blank">Results</a> are visualized using the enrichment map plug-in for Cytoscape. Each circular node is a gene set with diameter proportional to the number of genes. The outer node color represents the magnitude and direction of enrichment (see scale) in T47D cells, inner node color enrichment in MCF7 cells. Thickness of the edges (green lines) is proportional to the similarity of gene sets between linked nodes. The most related clusters are placed nearest to each other. The functions of prominent clusters are shown. The network can be examined in detail using the scalable PDF in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#pbio.1001461.s004" target="_blank">Figure S4</a>.</p