(A) Illustration of the ID4 gene promoter region and of MSP primer topology. Two CpG islands are located in the ID4 promoter region upstream of the transcription start site (TSS). Methylation-specific PCR (MSP) primers used for this study were positioned within the central CpG island (-615 until +139) near the TSS, detecting an amplicon indicated by the black box. The PCR product size of the unmethylated ID4 promoter sequence is 161 bp and similar in size to the product achieved with primers indicating methylated ID4 promoters (157 bp). The primers for the U reaction cover the bases -194 until -166 and -60 until -33. The primers for the M reaction cover the bases -192 until -166 and -60 until -35. All positions are relative to the TSS. (B) In vitro demethylation analysis of the ID4 promoter in four human breast cancer cell lines. Cells were incubated with 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA) for 72 h and 24 h, respectively. For each cell line, the methylation status and the fold change (FC) of ID4 mRNA re-expression are shown. All methylated cell lines (BT20, MCF7 and T47D) restored ID4 expression after demethylating treatment. MDA-MB231 remains unmethylated in the ID4 promoter and exhibits only a marginal increase of ID4 mRNA expression after DAC/TSA treatment. (C) ID4 promoter methylation analysis in primary human breast cancer (n = 170) using MSP technology. MSP results from nine representative patients (#) are shown. DNA bands in lanes labelled with U indicate PCR products amplified with primers recognising the unmethylated promoter sequence. DNA bands in lanes labelled with M represent products amplified with primers specific for methylated alleles. Human breast cancer cell lines BT20 and MDA-MB321 were used as positive controls for methylated and unmethylated ID4 promoter sequences, as described previously . Water was used as non template control (NTC).