Additional file 8: Figure S3. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism by BIMs2A slows tumor growth in mice bearing MDA-MB-468-2A xenografts but not MDA-MB-231-2A xenografts. (A–F) Line graphs depicting the tumor growth curves of MDA-MB-468-2A xenografts (A, B) and MDA-MB-231-2A xenografts (C, D) from mice fed with DOX or control food. Linear regression of these curves shown in B and D respectively. A comparison of the growth rate of tumors in mice bearing MDA-MB-468-2A (black) and MDA-MB-231-2A (red) xenografts fed with control food (E, F). (JPG 1348 kb

Additional file 5: Figure S5. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism resulted in changes in proteins involved in SRC family kinase signaling and phosphorylation at serine3 of Cofilin. (A) Normalized z-ratio (a measure of statistical significance) of phosphorylated and total proteins (as indicated) in MDA-MB-468-2A cells at 24 hours after treatment with DOX compared with control cells. (B) Western blots of serine 3 phosphorylated Cofilin, total Cofilin and Actin in xenografts of MDA-MB-468-2A and MDA-MB-231-2A fed DOX food as indicated. (C) Bar graphs depicting the ratio of serine 3 phosphorylated Cofilin to total Cofilin from (B). Bars indicate statistically significant groups, Mann–Whitney p value. (D) Immunofluorescence of Cofilin and p-Cofilin MDA-MB-231-2A cells grown on fibronectin 24 hours after DOX or vehicle treatment. (E) Proximity ligation assays using antibodies to MCL-1 and Cofilin (green), Phalloidin (red) and Dapi (blue) in MDA-MB-231-2A cells. (JPG 1419 kb

Additional file 6: Figure S6. of MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib

showing that MCL-1 antagonism and Dasatinib treatment induced apoptosis in MDA-MB-468-2A cells but not MDA-MB-231-2A cells when grown in 2D monolayer cultures. Bar graphs depicting the average fraction of apoptotic cells (total Annexin V-positive by flow cytometry) as indicated after 24 hours after treatment with vehicle, DOX, 5 μM A1210477 and 5 μM UMI-77 alone and in combination with 1 μM dasatinib after 24 hours. All graphs and western blots are the average of three independent experiments. Bars indicate statistically significant groups, p value paired t tests. (JPG 1029 kb

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

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

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

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