according to the protocol outlined in the Illumina technical manual. The arrays were scanned using Ilumina BeadArray Reader and analyzed using BeadStudio version 3.1.3.0. For each cell line, duplicates from two independent harvests were profiled. The absolute data was exported into GeneSpring GX v7.3 software (Silicon genetics) for further analysis. The measurements on each chip were first divided by the 50th percentile value, after which the value obtained for each gene was normalized to the average baseline median value of the control samples. One-way ANOVA approach was used to identify differentially-expressed genes and genes showing an average of at least a two-fold change in expression level in the four gelsolin-overexpressing samples. The output was then functionally annotated via Gene Ontology functional annotation under DAVID Bioinformation Resources (http://david.abcc.ncifcrf.gov/). The microarray data reported here are described in accordance with MIAME guidelines, and is accessible at Gene Expression Omnibus (GEO) database [64] through GEO Series accession number GSE36588 (http://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE36588). Real Time Quantitative RT-PCR Real Time PCR was used to validate gene expression change from microarray data. 0.5–1.4 mg of cDNA was used for reverse- transcription, carried out on the ABI 7300 Real time PCR system. The thermal cycling conditions were as follows: one cycle at 95uC for 10 minutes, followed by 40 cycles of denaturation at 95uC for 15 seconds and annealing extension at 60uC for 1 minute. The primers used included uPA (Hs01547054_m1), uPAR (Hs00182181_m1), PAI-1 (Hs00167155_m1), PAI-2 (Hs00234032_m1) and a2-antiplasmin (Hs00171467_m1). GAPDH (Hs99999905_m1) was included as the internal control. All reagents are from Applied Biosystems. Western Blotting Total cell lysates were extracted using lysis buffer (6 M urea, 1% SDS, 2 M b-mercaptoethanol, 1 M Tris pH 7.4, PBS). Equivalent amounts of protein from each sample were separated on 10% SDS-PAGE gels and transferred to PVDF membranes, blocked and incubated with primary antibody overnight at 4uC followed by secondary antibody before chemiluminescence substrate de- tection. Zymography Secreted uPA enzymatic activities were examined by zymo- graphy. Briefly, cells were grown in serum-free medium for 3 to 16 hours. The conditioned media were normalized using cellular lysate and then combined with non-reducing sample loading buffer and loaded into 10% SDS-PAGE gels containing 730 ug/ mL of human fibrinogen and 20 ug/mL human plasminogen (Sigma-aldrich). After electrophoresis, gels were rinsed with distilled water and incubated with wash buffer (2.5% Triton X- 100 and 50 mM Tris-HCl pH 8.0) for 1 hour, followed by incubation at 37uC with incubation buffer (0.1 M glycine buffer, pH 8.0) for 16 hours. The gels were then stained with Coomassie blue (0.05% Coomassie dye, 40% methanol, and 10% acetic acid) for 1 hour at room temperature, and destained with distilled water. Areas of lysis were expected to appear as zones of clearance against the Coomassie blue-stained background of undegraded substrate. Statistical Analysis Statistical analysis was performed using Student’s t-test except for IHC gelsolin expression analysis in which Mann-Whitney test Gelsolin Induces Cancer Invasion via uPA Cascade was used. Differences between sample means were considered statistically significant with P,0.05. Supporting Information Figure S1 Gelsolin immunohistochemistry in human colon tissues. An example of gelsolin staining in adjacent liver metastases section is shown here. Negative controls, including primary antibody exclusion and IgG isotype control were included to confirm the specificity of the gelsolin antibody used. Gelsolin was consistently highly expressed in the stroma but stromal stainings were mainly undetectable in the negative control samples. Bar: 50 mm. (TIF) Figure S2 Gelsolin immunohistochemistry in normal human colon tissues. Gelsolin is prominent in surface epithelium cells (A, arrowed) and weakly expressed, or absent in goblet cells (B, C). Cytoplasmic labelling is predominant, with occasional nuclear localization (C, arrowed). Gelsolin is intensely expressed in muscularis propria (D), vessel walls (E) and lymphoid cells (F). Bar: 50 mm. (TIF) Figure S3 Gelsolin immunohistochemistry in human colorectal carcinoma cell lines. In a panel of colorectal cell lines, gelsolin levels are highest in COLO201 and COLO205, both of which were obtained from metastatic ascites. Gelsolin expression in the remaining primary tumor-derived cell lines (except SW620 which was derived from lymph node) were more varied. The graph displays normalized gelsolin expression to HCT116. b-actin was used as loading control. (TIF) Figure S4 Cloned sequence of human cytoplasmic gelsolin cDNA. Nucleotides 159 to 2354 was cloned. This sequence is a 100% match with Genbank accession #BC026033.1. (TIF) Figure S5 Gelsolin modulates the expression of genes important for tumor dissemination. (A) The global gene expression profile of four stable gelsolin-overexpressing HCT116 cell lines were compared against the pooled average of two vector control cell lines using microarray analysis. Each horizontal row in the cluster diagram represents a gene. Blue shades represent downregulation while red shades represent upregulation in gene expression relative to the vector control cells. All samples were assayed in independent duplicates. (B) Biological function classification of differentially-expressed genes from the microarray output, using Gene Ontology annotation from DAVID bioinfor- mation resources. Genes showing an average of at least two-fold change in expression level in the four gelsolin-overexpressing clones are represented in the classification. Gelsolin modulates the genes involved in tumor dissemination including cell differentia- tion, cell motility and the regulation of cell adhesion. (TIF) Figure S6 Gelsolin-overexpressing HCT116 may en- hance invasion by MMP-2. Gelsolin-overexpressing cells were treated with MMP inhibitors and examined for changes in invasive potential through matrigel. The pan-MMP inhibitor GM6001 as well as inhibitors to MMP-2, MMP-2/-9 and MMP-3 were used at 40 mM, MMP-8 inhibitor at 1 mM, and a-MMP7 antibody at 80 mg/mL. DMSO and goat IgG antibody were used as controls to MMP chemical inhibitors and a-MMP7 antibody treatments respectively. No significant reduction in invasion was observed in PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e43594