Up-regulation of IL-1α mRNA expression after treatment with 5-aza-CdR. (A) Ms-SNuPE analysis of 4 representative CpGs (CpGs +1, –2, –7, and –8) in the IL-1α promoter in nonexpressing lymphoblastoid cell line Namalwa, the constitutive IL-1α–expressing cell line HaCaT, and CD4⁺ T cells. CpG –8 was always highly methylated and the other 3 CpGs were differentially methylated in the T-cell clones. The analyzed CpGs are indicated by the filled circles and black arrows in the schematic representation of the promoter region. The open circles indicate the CpGs not analyzed. (B) Expression of IL-1α in nonexpressing cell line Namalwa after treatment with 0.05 or 1 μM 5-aza-CdR. mRNA expression for IL-1α (35 cycles) and GAPDH (20 cycles) was examined by RT-PCR. The decrease in methylation indicated below each of the 5-aza-CdR concentrations used was quantitated by Ms-SnuPE and given as a percentage of the untreated sample. (C) Relative expression of the G and T allele in the predominantly G allele–expressing T-cell clones 41 and 90 that have been treated with (+) or without (–) 2 μM 5-aza-CdR and after a 6-hour stimulation with PMA and ionomycin. IL-1α mRNA levels were determined by real-time PCR and the contribution of each allele quantified by ASTQ. The contribution of each allele is represented as a percentage of the total with the G allele in gray and the T allele in white. Genomic DNA was analyzed by ASTQ as a control with an expected equal contribution of each allele.