E included in each plate. Relative expression values were obtained by

E included in each plate. Relative expression values were obtained by

E included in each plate. Relative expression values were obtained by the comparative Ct method [34].Statistical AnalysisDifferences in relative expression values of each gene in order MNS different groups were assessed by the Kruskall-Wallis non-parametric test, followed by pair-wise comparisons using the Mann-Whitney nonparametric test. The Chi-square test was used to assess the statistical significance of the differences in the frequency of methylation between NPT and PCa samples and a t-test was applied to qPCR and qMSP data. A p-value below 0.05 was considered statistically significant. The statistical analyses were performed using the Statistical Package for Social Sciences software, version 15.0 (SPSS Inc., Chicago, IL).Methylation-specific PCR (MSP) and Quantitative MSP (qMSP)To confirm the presence of a CpG island in the promoter region of the genes of interest, their RefSeqs were obtained from the USCS Genome Browser Database (http://genome. ucsc.edu/), including the 2 Kb sequence upstream of the first exon, and these were subsequently analyzed in silico using CpG Island Searcher software, according to the algorithm described by Takai and Jones (2002) [35]. The primers’ sequences for CAV1, IGFBP3, and LDOC1 have been published elsewhere [36?38] and the primers’ sequences for TGFBR2 and ECRG4 are shown in Supplementary Table S1, all being acquired from Metabion (Martinsried, Germany). MSP assays were carried on prostate samples using 2 mL of template modified-DNA in a 20 25837696 mL PCR reaction containing 0.2 mM of dNTPs mix (Fermentas, Ontario, Canada), 0.25 mM of each primer and 0.5 U of DyNAzymeTM II Hot Start (Finnzymes) in 1x DyNAzymeTM II Hot Start Reaction Buffer (ZK 36374 biological activity Finnzymes, Vantaa, Finland). PCR was then performed according to the DyNAzymeTM II Hot Start manufacturer’s conditions. Considering the limited amount of bisulfite-treated DNA available for the MSP analysis, samples were selected according to the lowest expression for each gene (14 for ECRG4, 10 for CAV1, eight for IGFBP3 and LDOC1 and seven for TGFBR2) (Supplementary Table S2). For qMSP on DAC-treated cell lines, 2 mL of bisulfite modifiedDNA were amplified with 0.25 mM of each primer in 16 Power SYBRH Green PCR Master Mix (Applied Biosystems). b-Actin (ACTB, Supplementary Table S1) was used as an internalResults Microarray Expression Data and Candidate Target Gene SelectionAfter crosschecking the list of EWSR1-FLI1 target genes in ESFT [20] with our microarray expression data on PCa and NPT, and applying the aforementioned selection criteria, seven potential ETS target genes emerged. Two genes were overexpressed in PCa with ERG fusion genes, namely HIST1H4L and KCNN2, and were chosen for validation. Five genes were underexpressed in PCa with ERG fusion genes, namely ABCD1, ECRG4, KCNMA1, LDOC1 and SLC7A4. ECRG4 and LDOC1 were selected for further analysis based on their putative function as tumor suppressor genes in other cancer types [43?4]. The expression of the selected target genes in Ewing’s sarcoma (CAV1, NR0B1, IGFBP3 and TGFBR2), together with the expression of HIST1H4L, KCNN2, ECRG4 and LDOC1, was then validated in an independent series of PCa with and without ETS gene fusions, as well as in a series of ESFT and ARMS.ETS Fusion Targets in CancerCAV1 Relative ExpressionCAV1 was significantly overexpressed in ESFT when compared to ARMS, showing a median 4.9 fold increase (Figure 1A). On the other hand, CAV1 was significantly underexpressed in PCa ETS+ when compared to P.E included in each plate. Relative expression values were obtained by the comparative Ct method [34].Statistical AnalysisDifferences in relative expression values of each gene in different groups were assessed by the Kruskall-Wallis non-parametric test, followed by pair-wise comparisons using the Mann-Whitney nonparametric test. The Chi-square test was used to assess the statistical significance of the differences in the frequency of methylation between NPT and PCa samples and a t-test was applied to qPCR and qMSP data. A p-value below 0.05 was considered statistically significant. The statistical analyses were performed using the Statistical Package for Social Sciences software, version 15.0 (SPSS Inc., Chicago, IL).Methylation-specific PCR (MSP) and Quantitative MSP (qMSP)To confirm the presence of a CpG island in the promoter region of the genes of interest, their RefSeqs were obtained from the USCS Genome Browser Database (http://genome. ucsc.edu/), including the 2 Kb sequence upstream of the first exon, and these were subsequently analyzed in silico using CpG Island Searcher software, according to the algorithm described by Takai and Jones (2002) [35]. The primers’ sequences for CAV1, IGFBP3, and LDOC1 have been published elsewhere [36?38] and the primers’ sequences for TGFBR2 and ECRG4 are shown in Supplementary Table S1, all being acquired from Metabion (Martinsried, Germany). MSP assays were carried on prostate samples using 2 mL of template modified-DNA in a 20 25837696 mL PCR reaction containing 0.2 mM of dNTPs mix (Fermentas, Ontario, Canada), 0.25 mM of each primer and 0.5 U of DyNAzymeTM II Hot Start (Finnzymes) in 1x DyNAzymeTM II Hot Start Reaction Buffer (Finnzymes, Vantaa, Finland). PCR was then performed according to the DyNAzymeTM II Hot Start manufacturer’s conditions. Considering the limited amount of bisulfite-treated DNA available for the MSP analysis, samples were selected according to the lowest expression for each gene (14 for ECRG4, 10 for CAV1, eight for IGFBP3 and LDOC1 and seven for TGFBR2) (Supplementary Table S2). For qMSP on DAC-treated cell lines, 2 mL of bisulfite modifiedDNA were amplified with 0.25 mM of each primer in 16 Power SYBRH Green PCR Master Mix (Applied Biosystems). b-Actin (ACTB, Supplementary Table S1) was used as an internalResults Microarray Expression Data and Candidate Target Gene SelectionAfter crosschecking the list of EWSR1-FLI1 target genes in ESFT [20] with our microarray expression data on PCa and NPT, and applying the aforementioned selection criteria, seven potential ETS target genes emerged. Two genes were overexpressed in PCa with ERG fusion genes, namely HIST1H4L and KCNN2, and were chosen for validation. Five genes were underexpressed in PCa with ERG fusion genes, namely ABCD1, ECRG4, KCNMA1, LDOC1 and SLC7A4. ECRG4 and LDOC1 were selected for further analysis based on their putative function as tumor suppressor genes in other cancer types [43?4]. The expression of the selected target genes in Ewing’s sarcoma (CAV1, NR0B1, IGFBP3 and TGFBR2), together with the expression of HIST1H4L, KCNN2, ECRG4 and LDOC1, was then validated in an independent series of PCa with and without ETS gene fusions, as well as in a series of ESFT and ARMS.ETS Fusion Targets in CancerCAV1 Relative ExpressionCAV1 was significantly overexpressed in ESFT when compared to ARMS, showing a median 4.9 fold increase (Figure 1A). On the other hand, CAV1 was significantly underexpressed in PCa ETS+ when compared to P.

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