Interaction of SPARC with TGFBI is necessary for TGFBI extracellular matrix deposition.

<p>(a) GST pull-down assay from SKOV3 cell lysates utilizing truncated GST-fusion proteins derived from the Carboxy-terminus of SPARC. Following GST-pull down, Western blot analysis was performed utilizing antibodies specific to the indicated proteins. (b) GST pull-down assay from SKOV3 cell lysates using either full-length SPARC or full-length SPARC lacking the carboxy-terminal 37 amino acids (aa 18–266). Following GST-pull-down (PD), Western blot analysis was performed to the indicated proteins. Coomassie brilliant blue staining of SDS-PAGE confirms expression and purification of GST fusion proteins. (c) Full-length SPARC-YFP or SPARC-YFP lacking the carboxy-terminal 47 amino acids (SPARC-YFP aa 1–256) were transiently transfected into Met5a cells and Western blot analysis was performed to confirm their expression. (d) and (e) Extracellular matrix preparation derived from these cells following 6-day culture was subsequently processed for confocal immunofluorescence microscopy. Cell derived ECM was immunostained for YFP, TGFBI, and SPARC as indicated. Merged images are indicated. Scale bar 40 μm.</p

TGFBI produced by mesothelial cells forms a fibrillar matrix distinct from fibronectin and loss of SPARC expression disrupts TGFBI deposition in mesothelial-derived ECM.

<p>(a) Western blot analysis of RIPA soluble lysates derived from a panel of ovarian cancer cell lines and the Met5a mesothelial cell line either untreated or treated with TGFß1. The membrane was probed with antibodies specific to the indicated proteins. (b) Confocal microscopy of Met5a cells and extracellular matrix preparations denuded of Met5a cells cultured for 9 days, was performed following immunostaining for TGFBI, fibronectin, Collagen type I, and Collagen type IV. Hoechst dye was utilized to visualize nuclei and merged images are indicated. Scale bar 40 μm. Quantitation of colocalisation from >3 fields of view were performed and represented as a Pearson’s coefficient. Error bars indicate s.d., ****p<0.0001, ***p<0.001. (c) RIPA soluble lysates harvested from Met5a cells stably expressing either control non-target shRNA or SPARC shRNA target #2 and #5, while cultured under 3D matrix conditions. Western blot analysis was performed utilizing antibodies specific to the indicated proteins. (d) Confocal microscopy of matrix preparations from Met5a cells stably expressing non-target shRNA, SPARC shRNA #2, or SPARC shRNA #5 immunostained for TGFBI and fibronectin. Merged images are indicated. Arrows indicate individual foci. Scale bar 200 μm. (e) Quantitation of results from immunostained matrices. The number of enriched TGFBI immunostained foci were counted from each group and represented as number of TGFBI foci/10X field of view. ***represents significance from control of p<0.001, ANOVA. (f) Conditioned media was harvested from Met5a cells stably expressing either control non-target shRNA or SPARC shRNA #5, while cultured under 3D matrix conditions. Western blot was performed utilizing antibodies specific to the indicated proteins.</p

Mesothelial-derived ECM influences cancer cell motility and response to the chemotherapeutic agent paclitaxel.

<p>(a) Time lapse video microscopy was performed of SKOV3 cells plated on Met5A derived ECM derived from cells expressing either control shRNA (Met5A matrix) or SPARC shRNA (Met5A matrix—SPARC). Images were collected for 10 hours and cell centroids were tracked using Volocity software. Circles represent tracking distance and velocity of each individual cell and black bars represent the mean ±S.E.M. (b) SKOV3 cells were plated on either plastic or Met5A derived ECM derived from cells expressing either control shRNA or SPARC shRNA (- SPARC). Cells were treated with 0.003 μM, 0.03 μM, or 0.3 μM paclitaxel for 30 hours prior to staining with FITC-Annexin V and propidium iodide before analyzing by flow cytometry. Three independent experiments were performed and the results are represented by percent of cells in early apoptosis (Annexin V +, PI -). ** represents significance of p<0.01 and *** represents significance of p<0.001.</p

SPARC directly interacts with TGFBI via its carboxy-terminus.

<p>(a) <i>In vitro</i> GST-binding assays. Coomassie stained gel of purified GST and GST-SPARC (aa 18–303) expressed in bacteria. Western blot analysis was performed following GST pull-down from SKOV3 lysates, probed with antibodies specific to the indicated proteins. (b) Coomassie stained gel of bacterially expressed and purified GST, GST-SPARC (aa 18–303), GST-SPARC Nterm (aa 18–134), and GST-SPARC Cterm (aa 154–303) fusion proteins. Western blot analysis was performed following GST pull-down assay from SKOV3 lysates, probed with antibodies specific to the indicated proteins. (c) <i>In vitro</i> binding of purified GST-SPARC to bacterially expressed and purified recombinant TGFBI. GST, GST-SPARC (aa 18–303), or GST-SPARC Nterm (aa 18–134) fusion proteins were incubated with rTGFBI or fibronectin, followed by pull-down with Glutathione sepharose 4B beads. Subsequently, Western blot analysis was performed with antibodies specific to the indicated proteins. Coomassie stained gel represents experimental input.</p

SPARC colocalizes with TGFBI in mesothelial-derived ECM.

<p>(a) Western blot analysis was performed on RIPA soluble lysates harvested from Met5a cells transfected with either YFP alone or SPARC-YFP. Immunoblotting with anti-GFP antibody recognizes YFP fusion constructs. (b) Extracellular matrix preparation was carried out from Met5a cells transfected with SPARC-YFP. Confocal microscopy was performed following immunostaining for YFP, TGFBI, fibronectin, and SPARC as indicated. Merged images are indicated. Scale bar 40 μm.</p

Application to a case of endometrioid cancer.

<p>A) Evolutionary tree of the OV03-04 case reconstructed from whole genome copy-number profiles. Approximate support values indicate how often each split was observed in trees reconstructed after resampling of the distance matrix with added truncated Gaussian noise. MEDICC performs reconstruction of ancestral copy-number profiles. Here, the (compressed) ancestral profiles for chromosome 17 are given as an example and MEDICC depicts unresolved ambiguities in the form of sequence logos. A star indicates no change compared to its ancestor. B) Ordination of the samples using kPCA shows four clear clonal expansions, comprising three separate Omentum groups and the Bl/VV group. C) Circos plot of selected genomic profiles (marked in bold in the tree) shows the extent of chromosomal aberrations across the genome. The two phased parental alleles are indicated in red and blue.</p

Efficient distance calculation is enabled via a transducer architecture.

<p>A) Overlapping genomic rearrangements modify the associated copy-number profiles in different ways. Amplifications are indicated in green, deletions in red. The blue rectangles indicate the previous event. B) The one-step minimum event transducer describes all possible edit operations achievable in one event. This FST is composed times with itself to create the the full minimum event FST . Edge labels consist of an input symbol, a colon and the corresponding output symbol, followed by a slash and the weight associated with taking that transition. C) The minimum event FST is asymmetric and describes the evolution of a genomic profile from its ancestor. Composed with its inverse this yields the symmetric minimum event distance .</p

Evolutionary copy-number trees are reconstructed in three steps.

<p>1) After segmentation and compression, major and minor alleles are phased using the minimum event criterion. 2) The tree topology is reconstructed from the pairwise distances between genomes. 3) Reconstruction of ancestral genomes yields the final branch lengths of the tree, which correspond to the number of events between genomes.</p

MEDICC improves reconstruction accuracy over competing methods.

<p>A) Simulations results show the improvement of reconstruction accuracy for MEDICC over naive methods (BioNJ clustering on Euclidean distances between copy-number profiles, red) and competing algorithms (TuMult, green). B) Allele phasing accuracy across the simulated trees. On average 92.9% of all genomic loci were correctly assigned to the individual parental alleles. C) Density estimates of clonal expansion indices for neutrally evolving trees (red) and trees with induced long branches as created by clonal expansion processes (blue) show the ability of MEDICC to detect clonal expansion.</p

Parental alleles are phased using context-free grammars.

<p>A) Allelic phasing is achieved by choosing consecutive segments from either the major or minor allele which minimise the pairwise distance between profiles. B) The set of all possible phasing choices is modelled by a context-free grammar. In this representation, the order of the regions' copy-number values on the second allele is reversed, in order to match the inside-out parsing scheme of CFGs. That way every possible parse tree of the grammar describes one possible phasing.</p