IGFBPs, ECM proteins, and other proteins detected in the CM of BC and MCF-10A cells.

<p>A-C. The number of SCs identified in the CM of each cell line is indicated. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. NSAF data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158296#pone.0158296.s003" target="_blank">S3 Table</a>. D. BC gene expression data was extracted from TCGA and boxplots were created for luminal A (pink) and TNBC (blue) tumors. Expression values are log2 normalized, tumor matched normal, with normal mammary tissue expression set to 0. (Abbreviations: CM = conditioned medium, BC = breast cancer, SC = spectral counts, TCGA = The Cancer Genome Atlas, TNBC = triple negative breast cancer)</p

Putative exosomal proteins detected in the CM of BC and MCF-10A cells.

<p>A. The number of SCs identified in the CM of each cell line is indicated. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. NSAF data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158296#pone.0158296.s003" target="_blank">S3 Table</a>. B. STRING analysis illustrates the numerous complex interactions possible among the putative exosomal proteins. Thicker lines reflect a higher confidence score. (Abbreviations: CM = conditioned medium, BC = breast cancer, SC = spectral counts)</p

Proteases detected in the CM of BC and MCF-10A cells.

<p>A. The number of SCs identified in the CM of each cell line is indicated. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. NSAF data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158296#pone.0158296.s003" target="_blank">S3 Table</a>. B. BC gene expression data was extracted from TCGA and boxplots were created for luminal A (pink) and TNBC (blue) tumors. Expression values are log2 normalized, tumor matched normal, with normal mammary tissue expression set to 0. C. Kaplan-Meier survival curves for human BC patients were created. The x axis represents the months of recurrence-free survival. Red and black curves indicate higher and lower expression, respectively. The p-value for each result is shown in the upper right quadrant. (Abbreviations: CM = conditioned medium, BC = breast cancer, SC = spectral counts, TCGA = The Cancer Genome Atlas, TNBC = triple negative breast cancer)</p

Identification of proteins in BC and MCF-10A cell secretomes.

<p>A. A workflow was established to analyze the secretomes of BC and benign mammary epithelial cells. B. Benign (MCF-10A), ERα-positive BC (MCF-7), and TNBC (MDA-MB-231, DT22, and DT28) cells were established in 3D cultures using Matrigel extracellular matrix. Live (top row) and fixed (bottom row) cultures were imaged by phase contrast or immunofluorescence, respectively. Markers of proliferation (Ki67-red), basement membrane (laminin-green), and nuclei (DAPI-blue) were utilized. C. 3D cultures of MCF-10A cells were exposed to CM from MCF-10A, MCF-7, MDA-MB-231, DT22, or DT28 cells. Proliferation (red) and nuclei (blue) are indicated by Ki67 and DAPI staining, respectively. Scale bars indicate 25 μm. D. 3D cultures described in panel C were analyzed to determine the percent of proliferating MCF-10A cells for each treatment. An asterisk (*) indicates a p-value <0.05. E. CM from each cell line was subjected to MS and compiled results are shown. F. CM from 3D cultures of BC and MCF-10A cells were subjected to Western blotting analysis using antibodies to cathepsin D (CTSD), extracellular matrix protein 1 (ECM1), peroxiredoxin 1 (PRDX1), or 14-3-3 sigma (SFN). The number of spectral counts (SCs) is indicated above each lane. To visualize SFN, the CM was concentrated before blotting. MDA-MB-231 and MCF-10A cells are labeled as 231 and 10A, respectively. (Abbreviations: BC = breast cancer, TNBC = triple negative breast cancer, 3D = three dimensional, CM = conditioned medium, MS = mass spectroscopy)</p

Presence of predicted orthologs of SMCL-1, DPY-27 (I^DC), and SMC-4 (I & II) in various species.

<p>Phylogenetic tree built from all available <i>Caenorhabditis</i> species with sequenced and well-assembled genomes, other selected nematode species, and other selected model organisms. “<i>+”</i> symbol denotes the presence of SMCL-1-like protein based on similarity in a BLAST search and the additional criteria of short length, imperfect signature motif, and a Walker B motif lacking the catalytic glutamate (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#sec014" target="_blank">Methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s003" target="_blank">S3 Fig</a>). “1” denotes orthologs detected using a high-confidence Ensemble-COMPARA method and “2” denotes orthologs detected using BLAST-neighbor-joining tree methods.</p

Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

<div><p>The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.</p></div

Condensin subunits co-purify with MAP::SMCL-1.

<p>(A-B) Proteins that co-purified with MAP::SMCL-1 but not untagged control adult extracts, identified by tandem affinity purification and MudPIT mass spectrometry. Numbers represent average NSAF values from two replicas. Co-purified proteins with the highest NSAF values are shown in (A), values for other condensin subunits are shown in (B), and all other proteins are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s011" target="_blank">S5 Table</a>. Condensin SMC subunits are highlighted.</p

SMCL-1 expression and protein features.

<p>(A) Adult hermaphrodites from wild-type (WT) and a strain carrying the <i>map</i>::<i>smcl-1</i> transgene driven by endogenous <i>smcl-1</i> 5’ and 3’ elements. A section of the germline is shown, imaged by DIC to show structures and fluorescent microscopy to detect mVenus expression from the MAP tag. Arrowheads denote the first four oocytes. (B) A typical SMC protein folds back on itself at a hinge domain, bringing coil regions together and creating a “head domain” (yellow) from ATPase domains in the N- and C-termini. SMCL-1 lacks predicted coil and hinge domains, but has N- and C-terminal ATPase domains that may be capable of forming a head domain (purple). (C) SMC head domain and the ATPase cycle, showing binding of ATP (red circle), ATP-dependent engagement of heads from two SMC proteins, and disengagement upon ATP hydrolysis. (D) SMCL-1 amino acid sequence aligned to <i>C</i>. <i>elegans</i> condensin SMC proteins. Shown are regions surrounding three conserved motifs found in SMCs and related ATPases: the Walker A motif, ABC transporter signature motif, and Walker B motifs, and their consensus sequences. SMCL-1 shares a conserved Walker A motif, but differs from consensus signature motif and Walker B motif at residues shown in red. Asterisk denotes catalytic amino acid required for ATP hydrolysis. x = any amino acid and h = hydrophobic amino acid.</p

RT-PCR demonstrates the quantitative nature of the MS data.

<p>RNA was isolated from each of the cell lines, cDNA was made, and RT-PCR was performed to determine whether the spectral ID numbers were correlated to transcript levels of selected genes. Spectral ID numbers are displayed above each graph and the gene symbol is below those numbers.</p

SMCL-1 overexpression in the gut disrupts condensin I^DC localization on the X chromosomes.

<p>(A) Heat shock regimen for data in (B-E). Bolt represents the single heat-shock pulse given to young adult hermaphrodites from the wild-type or inducible <i>hs</i>:<i>smcl-1(+)</i> transgenic strain. (B-E) Adult hermaphrodite gut tissue of the indicated strain and treatment was stained with DAPI to image DNA (green in merge) and immuno-stained with antibody against CAPG-1(B-C), DPY-27 (D) and DPY-28 (E), (red in merges). Antibody against SMCL-1 was also included in (B and C), showing overexpression upon heat shock (blue in merge). The foci of staining created by condensin I^DC subunit association with the X chromosomes are lost when SMCL-1 is overexpressed. (C) A mosaic animal in which anti-SMCL-1 staining indicates that one cell lacks the transgene (left) and shows foci of anti-CAPG-1, while a neighboring cells has the <i>smcl-1</i> overexpression transgene (right) and CAPG-1 staining is weak and not localized to foci (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006614#pgen.1006614.s006" target="_blank">S6C and S6D Fig</a>). HS = heat shock.</p