5-Azacytidine relieves AML1/ETO transcriptional silencing of the RA signaling pathway and supports RA-induced differentiation. (A) The bisulfite sequencing assay was performed on genomic DNA isolated from SKNO-1 cells and SKNO-1 –infected (siA/E) cells. Cells were treated or not with 5-azacytidine (1 μM) for 48 hours, and then RA (1 μM) was added for an additional 48 hours in the indicated samples. The methylation status of each CpG from nt −370 to +238 of the RARβ2 promoter/exon 1 DNA sequence was measured and represented by a circle depicted by increasing gray intensities. The increasing gray-intensity scale indicates a 10% rise in methylation status of the CpG of interest and summarizes the results of the analysis of 6 different clones for each sample. For each sample, the percentages of global methylation level in the promoter and in the 5′-UTR exon 1 regions are indicated. (B) SKNO-1 cells were treated or not with 5-azacytidine (1 μM), RA (1 μM), or in combination for 48 hours as described. The qRT-PCR results of RARβ2 mRNA expression and the percentage of CD11b⁺ SKNO-1 cells measured by FACS analysis are shown. The results represent an average of 3 independent evaluations ± SD.