Promoter CpG island Hypermethylation leads to repression of GITR in MM cells.

A) Primer location for Medip, MSP and bisulfite sequencing assay. Bar arrow represents 1 κB region on the genome. TSS represents transcription starting site. B) Analysis of DNA methylation status of 7 TNFRSF members promoter CpG islands in 5 MM cell lines. Methylated DNAimmunoprecipitation assay was performed to profile promoter methylation status. Promoter of housekeeping gene GAPDH was used as unmethylated control. Fold enrichment was calculated as described in the method and normalized to GAPDH. C) Bisulfite sequencing at the CpG island of GITR promoter, with circles denoting the methylation status of each selected clone. Black and white circles are methylated CG dinucleotides, and non-methylated CpG dinucleotides and TG dinucleotides, respectively. D) Re-expression of GITR mRNA level was examined by real-time PCR in the presence of 5’ azacytidine (5 µM) for 4 days. Total RNA was extracted and reverse transcripted with oligo_dT. The mRNA level of GITR was normalized to 18s. Mean±SD. E) Expression of GITR was determined in 5 MM cell lines by real-time PCR. The mRNA level was normalized to 18s. Mean±SD. DNA methylation status of GITR in 5 MM cell lines was calculated by counting the methylated CG sites in the promoter of GITR and divided by total CG number. F) Expression of GITR examined in 9 primary MM and 11 normal bone marrow by tissue array using anti-human GITR monoclonal antibody. 4 slides of each group were shown. GITR expression was represented as brown color. Staining of tonsil for GITR staining was considered as positive control.