Quantitative Methylation Assay: 1 Μg of Genomic DNA Was Digested with the Methylation Sensitive

Supplemental Legends

Supplemental figure S1: Global demethylation of Hodgkin cell lines is unable to restore their B-cell phenotype. Expression of B-cell specific/characteristic genes in 5-aza-dC treated (+) and untreated (-) Hodgkin cell lines (L428, KMH2, L1236) and untreated (-) B-cell lines (Raji, Namalwa, Daudi, SU-DHL-4, SU-DHL-6). (A) Relative mRNA expression of B-cell specific genes as analyzed by means of real-time RT-PCR. B) Western blot analysis of B-cell specific proteins in 5-aza-dC treated (+) and untreated (-) Hodgkin cell lines.The data for each protein derive from the same gel (same exposure and adjustment of brightness).

Supplemental figure S2: Combined 5-aza-dC/TSA treatment is more effective than the treatment with 5-aza-dC and TSA alone: Relative mRNA expression of B-cell specific (A) and Hodgkin characteristic (B) genes in untreated (-), 5-aza-dC (A), TSA (T) and combined 5-aza-dC/TSA (A/T) treated Raji cell line cells.

Suupplemental figure S3: The extinction of the B-cell phenotype by combined global DNA-demethylation and histone-acetylation is confirmed by real-time RT-PCR.Relative mRNA expression of B-cell specific genes in 5-aza-dC/TSA treated (+) and untreated (-) B-cell lines determined by real-time RT-PCR.

Supplemetal figure S4: Combined global DNA-demethylation and histone-acetylation of B-cell lines leads to the up-regulation of B-cell inappropriate genes characteristically expressed in Hodgkin cells. Relative mRNA expression of Hodgkin-characteristic genes in untreated Hodgkin cell lines and 5-aza-dC/TSA treated (+) and untreated (-) B-cell linesanalyzed by real-time RT-PCR.

Supplemetal figure S5:Promoter histone (H3) acetylation status of B-cell and Hodgkin-characteristic genes analyzed by chromatin immunoprecipitation (ChIP). Relative enrichment of histone-acetylated promoter DNA regions after ChIP quantified by real-time DNA-PCR. Empty bars: input control DNA; grey bars: relative promoter H3 acetylation enrichment in comparison to input control DNA.(A)B-cell specific genes(B) Hodgkin characteristic genes

Supplemental figure S6: Promoter DNA-methylation status of Syndecan 4 by methylation sensitive restriction. Relative amplifiability of a promoter DNA region of Syndecan 4 after HpaII (methylation sensitive) and MspI (methylation unsensitive) digestion.

Supplemental figure S7: Variations of 5-aza-dC concentration or incubation time without significant influence on the gene expression pattern. Expression of B-cell specific/characteristic genes in 5-aza-dC treated (+) and untreated (-) Hodgkin cell lines (L428, KMH2, L1236) and untreated (-) B-cell lines (Raji, Namalwa, Daudi). Therelative mRNA expression of POU2AF1 (Bob1), CD19 and SYKwas analyzed by means of real-time RT-PCR. Hodgkin derived cell lines were treated with 5-aza-dC at a concentration of 5 µM for 5 days(A)or alternatively with 1 µM 5-aza-dC for 24 hours(B).The left part of (A) and (B) representsthe Hodgkin cell line data using an enlarged scale.