Y selected microscopic Beclabuvir web high-power fields (100?.Analysis of the TCGA dataMethylation status of ADAMST19 CGI was downloaded from the methHC webserver [63]. Methylation data of the TCGA ovarian cancer dataset is not included in methHC because most cases have been analyzed with the HM27K platform. Hence, this dataset was directly downloaded from the TCGA. COAD (colon) and READ (rectum) datasets were combined into a single dataset, representing colorectal carcinomas (CRC).Statistical analysisCell migration was assayed by two complementary methods: scratch/wound healing assays and Transwell plates (8 m pore size) (Millicell, Millipore). For the scratch/wound healing assays, cells were cultured until reaching confluency and then four different scratches were done using a P200 micropipette tip. The width (in ) of the scratches was measured at different positions using an automatized-capture Leica DMI 6000 B microscope. After 24 h of incubation, the width was measured again at the same coordinates that were previously stored in the microscope managing software. The collective migration speed was estimated dividing the difference in the scratch width by two, and then by 24h (see equation in figure S7). Clumps or colonies of cells inside the scratch but disconnected from the borders of the scratch were ignored. The experiments were performed in duplicate. For the Transwell plate assays, the undersurface PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 of the membrane was coated at 4 overnight with 40 g/mL of Collagen I (BD Bioscience, cat N. 354236) diluted in PBS and then blocked with 2 (w/v) BSA at room temperature for 2 h. The upper compartment was seeded with 2 ?105 o/n starved transfected cells per well in 200 L of serum-free DMEM + 0.5 BSA. DMEM + FBS (10 ) was added in the lower chamber. Cells were allowed to migrate through the membrane for 21 h. Cells capable of migrating through the membrane were stained with 0.5 (w/v) Crystal violet (Sigma) in 10 (v/v) ethanol. Each experiment used quadruplicate wells and, within each well,Statistical analyses were performed using the R statistical environment [64]. Association between two categorical variables was analyzed by Fisher’s exact test (for 2 ?2 contingency tables) or chi-square test (for larger contingency tables). Normality of continuous variables was assessed using the Shapiro-Wilk test. Comparisons between two groups were performed with the Student’s t test for variables following a normal distribution or with the nonparametric Mann-Whitney-Wilcoxon test for variables that do not follow a normal distribution. When more than two groups were analyzed, we applied ANOVA or rANOVA analyses followed by Tukey’s honest significant difference method. Trend analysis of categorical data was performed using the Cochran-Armitage test. The level of statistical significance was set at p < 0.01, unless otherwise specified. Holm's multi-hypothesis testing correction was applied when appropriate [65].Additional fileAdditional file 1: Figures S1? and Table S1. Validation of the methylation alterations in ADAMTS19, array CGH analysis of Chr5, association of ADAMTS19 with clinicopathological parameters, methylation and expression of ADAMTS19 in CRC cell lines, silencing of ADAMTS19 expression using shRNA constructs, effect of ADAMTS19-silencing on growth rate and anchorage free growth, effect of ADAMTS19-silencing on collective cell migration speed, and sequence of primers used in this study.Alonso et al. Clinical Epigenetics (2015) 7:Page 14 of.