Cas proteins activate SRC to downregulate E-cadherin expression.

<p><b>A</b>, <b>B</b>. Western analysis (<b>A</b>) of whole cell lysates prepared from MCF-7 cells overexpressing pcDNA vector alone (-), or HA-tagged BCAR1 (B), NEDD9 (N), or both (NB), and treated with dasatinib versus vehicle. Quantification of results comparing levels of E-cadherin, normalized to β-actin, within treated and, separately, untreated groups from 4 independent experiments, is also shown (<b>B</b>). Src Y⁴¹⁶ phosphorylation reflects activity state of kinase. P values reflect the difference between the vehicle and drug treatment condition, for each transfected protein indicated; *, P<0.01, **, P<0.001, ns, non-significant. <b>C</b>. Experiment as in <b>A</b> performed with PP2. Note, SRC inhibition was not as complete as in <b>A</b> in these experiments, because at higher PP2 concentrations extensive cell death was observed, probably due to the broader spectrum of PP2 versus dasatinib targets. <b>D</b>, <b>E</b>. Experiment as in <b>A</b>, <b>B</b>, but in cells treated with Y27632 versus vehicle. Phosphorylation of the downstream p160ROCK target MYPT1 was assessed in parallel to confirm complete inhibition of p160ROCK (not shown). *, P<0.001, **, P<0.01 ***, P<0.0001, ns, non-significant. Error bars represent SE.</p

Cas proteins negatively regulate association of E-cadherin with cell junctions.

<p><b>A</b>. qRT-PCR of mRNA isolated from MCF7 cells transfected with plasmids including vector pcDNA-HA (-), pcDNA-HA-NEDD9 (N), pcDNA-HA-BCAR1 (B), or pcDNA-HA-NEDD9 and pcDNA-HA-BCAR1 (NB). <b>B</b>. MCF7 transfected with siRNAs including scrambled control (SCR), or targeting NEDD9 (siN), BCAR1 (siB) and both (siNB), were stained for E-cadherin (red), and DAPI (blue). <b>C</b>. MCF7 cells transfected as in <b>A</b> and stained for E-cadherin (red), HA-fused proteins (green) and DAPI (blue). Arrows indicate cell-cell contacts of transfected cells. <b>D</b>. MCF7 cells transfected with plasmids including vector pcDNA-GFP, pcDNA-GFP-NEDD9, pcDNA-GFP-BCAR1, or pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (green) were stained for β-catenin (red), and DAPI (blue). Arrowheads point to β-catenin localization at the focal adhesions. Scale bar in <b>B–D</b>, 20 µm. <b>E</b>. Immunofluorescence demonstrating that β-catenin localizes to focal adhesions in MCF7 cells expressing pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (green). β-catenin (red) and the focal adhesion protein paxillin (blue) are indicated. Scale bar, 20 µm. Bottom panel represents magnifications of indicated areas from boxes.</p

Cas proteins negatively regulate E-cadherin expression in MCF7 cells.

<p><b>A</b>. Western analysis of MCF7 cells transfected with plasmids including vector pcDNA-HA (-), pcDNA-HA-NEDD9 (N), pcDNA-HA-BCAR1 (B), or pcDNA-HA-NEDD9 and pcDNA-HA-BCAR1 (NB). <b>B</b>. Western analysis of MCF7 transfected with siRNAs including scrambled control (scr), or targeting NEDD9 (siN), BCAR1 (siB) and both (siNB), probed with antibodies indicated. <b>C</b>. Graph represents total levels of E-cadherin normalized to β-actin. *, P<0.01, **, P<0.001. Error bars represent SE. Additional siRNA experiments (not shown) were performed with alternative siRNA oligonucleotides targeting NEDD9 and BCAR1: although knockdown was not as efficient, qualitatively similar results were obtained in regard to E-cadherin expression.</p

Fractionation indicates Cas proteins decrease E-cadherin association with the insoluble fraction.

<p>MCF7 cells were transfected as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022102#pone-0022102-g002" target="_blank"><b>Figure 2A and B</b></a>, fractionated to insoluble (<b>A</b>) and soluble (<b>B</b>) fractions, and then analyzed for expression of E-cadherin. Graphs represent E-cadherin normalized to β-actin in each fraction calculated from 3 independent experiments. Immunoblot represents a typical experiment. *, P<0.05, **, P<0.01, ***, P<0.001. Error bars represent SE.</p

Cas proteins increase lysosomal colocalization of E-cadherin.

<p><b>A</b>. Immunofluorescence assessing E-cadherin localization, with lysosomes visualized with LAMP-1 (green) in cells overexpressing GFP-BCAR1 (B) and GFP-NEDD9 (N), indicated in blue, after treatment with cloroquine. E-cadherin is shown in red. Scale bar, 20 µm. Arrows indicate colocalized E-cadherin and LAMP-1. <b>B–E</b>. Quantification of western analysis and representative results of whole cell lysates prepared from MCF-7 cells transfected with either the vector pcDNA-GFP, pcDNA-HA-NEDD9 (N), pcDNA-HA-BCAR1 (B), or pcDNA-HA-NEDD9 and pcDNA-HA-BCAR1 (NB), and treated with <b>B</b>. vehicle (DMSO), <b>C</b>. ammonium chloride (NH₃Cl), <b>D</b>. monensin or <b>E</b>. chloroquine, to inhibit lysosomal action. Graphs represents total levels of E-cadherin normalized to β-actin, calculated from more than 4 independent experiments. P values were calculated to compare levels of E-cadherin in vector-transfected cells versus cells transfected with NEDD9, BCAR1 or both within each drug treatment group; *, P<0.05, **, P<0.01, n.s., non-significant. Error bars represent SE.</p

Nedd9<i>^−/−</i> mammary tumors have increased E-cadherin at cell junctions.

<p><b>A</b>, <b>B</b>. Immunohistochemical evaluation of MMTV-PyVT;<i>Nedd9^−/−</i> and MMTV-PyVT <i>Nedd9^+/+</i> mammary tumors for E-cadherin (<b>A</b>) and β-catenin (<b>B</b>) protein expression and localization. Insets (thick lines) are magnifications of indicated areas (thin lines). Three representative independently arising tumors are shown for each genotype. Scale bar, 100 µm. <b>C</b>. Western analysis of total lysates isolated from 9 independent MMTV-PyVT;<i>Nedd9^−/−</i> and MMTV-PyVT <i>Nedd9^+/+</i> mammary tumors and from MCF7 cells were probed for content of E-cadherin and β-catenin. β-actin served as loading control.</p

Treatment with SRC inhibitors reverses CAS-induced downregulation of E-cadherin.

<p>Immunofluorescence of MCF7 cells expressing vector pcDNA-GFP, pcDNA-GFP-NEDD9, pcDNA-GFP-BCAR1, or pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (transfected cells shown in green) and treated with vehicle or PP2. Arrows indicate presence of E-cadherin (shown in red) at cell junctions in dasatinib or PP2-treated cells expressing vector, BCAR1 and/or NEDD9, but absence of E-cadherin in similarly transfected cells treated with vehicle. Scale bar, 20 µm.</p

Drug combination using dasatinib and CX-4945.

<p><b>A.</b> The Chou-Talalay method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref078" target="_blank">78</a>] was used to perform drug combination studies of dasatinib and CX-4945. The points represent the average viability ± standard error of mean following 72 h of drug treatment at the indicated concentrations of CX-4945 (•) and CX-4945 + dasatinib (◆; constant molar ratio of 20:1 of CX-4945:dasatinib) for the various EOC cell lines as a percentage of vehicle treated cells. The curve-fit lines were generated using non-linear regression analysis in GraphPad Prism. Data for the other molar ratios that were evaluated are presented in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s002" target="_blank">S2 Fig</a>. B.</b> The dose response data were used to calculate the Combination Index (CI) values for each cell line at the various molar ratios using CalcuSyn software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.ref079" target="_blank">79</a>]. CI values less than 1 suggest that the drugs are working synergistically. Shown is the average calculated CI value ± standard error of the mean.</p

Plk1 is activated during ciliary disassembly but has limited influence on disassembly dynamics.

<p><b>A</b> Immuno blot analysis of whole cell lysates prepared from hTERT-RPE1 grown under starved, ciliated conditions (0h) or at the times indicated following serum treatment to induce ciliary resorption. Ph-Plk1^T210 represents activated Plk1; <b>B, C</b> Quantification of the expression of (<b>B</b>) Ph-Plk1^T210 or (<b>C)</b> total Plk1 normalized to β-actin and relativized to the expression level without serum induction. Results from three independent experiments were calculated. * P<0.05, ***P≤0.005. Error bars represent SE. <b>D</b> Upper panel: Immunofluorescence of starved ciliated hTERT-RPE1 cells treated with the Plk1-inhibitor BI 2536 or with DMSO for 3 hours, then maintained in serum-free medium, or induced for ciliary disassembly with 10% serum-containing medium. Lower panel: Immunofluorescence of serum-starved, ciliated hTERT-RPE1 cells treated with siRNA to Plk1 or with scrambled (scr) control siRNA after 0, 2 and 24 hours of serum addition. Image shows acetylated α-tubulin (green), γ-tubulin (red) and DNA (blue). The scale bar represents 10 µm. <b>E</b> - <b>H</b> Quantification of three independent repetitions of experiment presented in <b>D</b>. <b>E</b> and <b>G</b> Quantification of the percentage of ciliated cells, with an average of 250 cells counted for each condition. <b>F</b> and <b>H</b> Quantification of cilia length, with an average of 50 cilia measured for each condition. * P<0.05, **P<0.01. ***P<0.001. Error bars represent SE.</p

Gene expression in clinical samples.

<p>Agilent gene expression data from TCGA on 518 serous cystadenocarcinomas and 8 fallopian tube samples derived from healthy individuals were queried for 29 dasatinib sensitizing genes. The six Agilent probes that showed ≥ 1.5-fold increase in the average gene expression of the respective genes in the tumor samples (gray boxes) relative to the controls (white boxes) are shown. The whiskers of each box plot represent the expression values at the 5^th and the 95^th percentiles. The p-values were calculated using an unpaired two-tailed t-test using GraphPad Prism. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144126#pone.0144126.s007" target="_blank">S4 Table</a></b> lists the average expression values of the Agilent probes across the tumor and normal samples for all 29 genes.</p