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Suppl. Fig. 1. Average Dm values for duplicate MS-MLPA analyses of the ovarian and endometrial cancer cell lines studied. HEC59, AN3CA, and ES2 exhibit.

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Presentation on theme: "Suppl. Fig. 1. Average Dm values for duplicate MS-MLPA analyses of the ovarian and endometrial cancer cell lines studied. HEC59, AN3CA, and ES2 exhibit."— Presentation transcript:

1 Suppl. Fig. 1. Average Dm values for duplicate MS-MLPA analyses of the ovarian and endometrial cancer cell lines studied. HEC59, AN3CA, and ES2 exhibit increased methylation at multiple loci which is compatible with clinical specimens of non-serous ovarian cancer (Fig. 2). The less frequent occurrence of methylation in CAOV3 is in agreement with serous ovarian cancers (Fig. 2). In contrast, the strong methylator phenotype in SKOV3 deviates from the generally low methylation frequencies observed in serous ovarian cancers and provides additional evidence to support the notion that the molecular features of SKOV3 are untypical of serous ovarian cancer (Domcke et al. Nat Commun 2013; 4:2126).

2 5600 2394 1715 Non-serous ovarian and endometrial cancer cell lines (ES2, HEC59 and AN3CA ) Serous ovarian cancer cell lines (CaOV3 and SKOV3) Suppl. Fig. 2 Suppl. Fig. 2. Venn diagram showing the number of mRNA probe sets with significant upregulation in one or several of the non-serous and serous cancer cell lines, as well as those with shared upregulation in at least one cell line from both groups (bold).

3 Bisulphite sequencing primers F R CpG sites CpG sites Normal uterus HEC59 SKOV3 LIM1215 HhaI sites in MS- MLPA probe Direct bisulphite sequencing color code = C/C = C/T = T/T bp A B Normal colon HCT116 ES2 Proportion of methylated DNA MS-MLPA Cloning 1.02 0.26 0.49 0.04 0.05 0.91 0.25 0.40 -- 0.07 0.86 0.22 Suppl. Fig. 3

4 Suppl. Fig. 3. A. The design and validation of MS-MLPA assays using SFRP2 as an example (see Materials and Methods for details). Bisulfite-converted DNA from cancer cell lines and normal tissues was first sequenced to select a representative region for MS-MLPA probe design. Results from direct bisulphite sequencing (without cloning) are shown on the left, together with proportions of methylated DNA assessed by two parallel methods, MS-MLPA and sequencing of cloned bisulfite-converted PCR-amplified fragments shown on the right. An average of 18 clones per cell line was analyzed to determine the proportion of methylated DNA by the cloning method. Dm values by MS-MLPA are concordant with results from the cloning experiments. B. The electropherograms for LS-associated endometrioid ovarian carcinoma (bottom) and normal unrelated endometrium (top) to depict a typical output from the custom-made MS-MLPA test. Dm values obtained for each locus are given below the name of the gene. Control peaks are indicated by arrowheads (the respective probes have no HhaI sites and are not affected by methylation). While most of the test loci are unmethylated in normal endometrium (virtually no MS-MLPA peak), the X-chromosomal RSK4 shows some methylation [Dm = 0.21 in the normal endometrium sample shown and 25-59% methylation reported in Dewdney et al. (Clin Cancer Res 2011; 17:2120-2129)]. Compared to the normal tissue reference (cut-off values given in Suppl. Table 4), the tumor sample shows hypermethylation at RSK4, SPARC, HOXA10, and HOXA9.

5 RSK4 200bp BS-R * MS-MLPA probe BS-F Exon 1 [Dewdney SB, Rimel BJ, Thaker PH, Thompson DM,Jr, Schmidt A, Huettner P, Mutch DG, Gao F, Goodfellow PJ. Aberrant methylation of the X-linked ribosomal S6 kinase RPS6KA6 (RSK4) in endometrial cancers. Clin Cancer Res 2011; 17:2120-2129.] Suppl. Fig. 4

6 [Rodriguez-Jimenez FJ, Caldes T, Iniesta P, Vidart JA, Garcia-Asenjo JL, Benito M. Overexpression of SPARC protein contrasts with its transcriptional silencing by aberrant hypermethylation of SPARC CpG-rich region in endometrial carcinoma. Oncol Rep 2007; 17:1301-1307.] SPARC 200bp BS-R * MS-MLPA probe BS-F Exon2

7 PROM1 200bp BS-F BS-R Promoter 2 * MS-MLPA probe Exon 2 [Baba T, Convery PA, Matsumura N, Whitaker RS, Kondoh E, Perry T, Huang Z, Bentley RC, Mori S, Fujii S, Marks JR, Berchuck A, Murphy SK. Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells. Oncogene 2009; 28:209-218.]

8 WT1 200bp BS-FBS-R * MS-MLPA probe [Kaneuchi M, Sasaki M, Tanaka Y, Shiina H, Yamada H, Yamamoto R, Sakuragi N, Enokida H, Verma M, Dahiya R. WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma. Cancer 2005; 104:1924-1930.] Exon 1

9 CABLES1 200bp MS-MLPA probe * BS-F BS-R Exon 1 [Sakamoto H, Friel AM, Wood AW, Guo L, Ilic A, Seiden MV, Chung DC, Lynch MP, Serikawa T, Munro E, Oliva E, Orsulic S, Kirley SD, Foster R, Zukerberg LR, Rueda BR. Mechanisms of cables 1 gene inactivation in human ovarian cancer development. Cancer Biol Ther 2008; 7:180-188.]

10 HOXA10 200bp ** BS-F BS-R MS-MLPA probe Exon 1 [Wu Y, Halverson G, Basir Z, Strawn E, Yan P, Guo SW. Aberrant methylation at HOXA10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis. Am J Obstet Gynecol 2005; 193:371-380.]

11 HOXA9 200bp * BS-FBS-R MS-MLPA probe Exon 1

12 WT1-AS 200bp * Promoter of WT1-AS BS-F BS-R MS-MLPA probe [Kaneuchi M, Sasaki M, Tanaka Y et al. WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma. Cancer 2005; 104:1924-1930.] WT1 exon 1

13 SFRP2 200bp * BS-F BS-R MS-MLPA probe Exon 1 [Cheng YY, Yu J, Wong YP, Man EP, To KF, Jin VX, Li J, Tao Q, Sung JJ, Chan FK, Leung WK. Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer. Br J Cancer 2007; 97:895-901.]

14 OPCML 200bp MS-MLPA probe * BS-F BS-R Exon 1 [Cui Y, Ying Y, van Hasselt A, Ng KM, Yu J, Zhang Q, Jin J, Liu D, Rhim JS, Rha SY, Loyo M, Chan AT, Srivastava G, Tsao GS, Sellar GC, Sung JJ, Sidransky D, Tao Q. OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation. Plos One. 2008; 3:e2990.]

15 SFRP5 200bp BS-F ** MS-MLPA probe BS-R Exon 1 [Cheng YY, Yu J, Wong YP, Man EP, To KF, Jin VX, Li J, Tao Q, Sung JJ, Chan FK, Leung WK. Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer. Br J Cancer 2007; 97:895-901.]

16 MIR34B 200bp BS-F MS-MLPA probe BS-R MIR34B * Toyota M, Suzuki H, Sasaki Y, Maruyama R, Imai K, Shinomura Y, Tokino T. Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer. Cancer Res. 2008; 68:4123-4132.

17 Let-7a-3 200bp MS-MLPA probe ** BS-FBS-R [Brueckner B, Stresemann C, Kuner R, Mund C, Musch T, Meister M, Sültmann H, Lyko F. The human let-7a-3 locus contains an epigenetically regulated microRNA gene with oncogenic function. Cancer Res. 2007; 67:1419-1423.] Let-7a-3 Suppl. Fig. 4. Structural characteristics of promoter-associated CpG islands for the genes investigated and the location of sequences for bisulfite sequencing (BS-F and BS-R) and MS-MLPA probes. Asterix denotes HhaI site. CpG islands are shown as blue bars. The first coding exon is depicted as a separate box consisting of untranslated (white) and translated (grey) portions. The initiating ATG is located at the start of the grey portion. References for selected publications with evidence of the chosen regions being functional targets of methylation are also given.


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