Methylation status of CpGs along the RARβ2 promoter/exon 1 regions in human primary AML blasts. (A) Methylation-sensitive PCR (MSP) assay performed to detect the methylation status of CpGs along the RARβ2 promoter/exon 1 regions on bisulfite-treated genomic DNAs from 19 primary AML blasts at diagnosis (9 cases of AML-M2, 10 cases of AML-M4 according to FAB classification) and CD34⁺ hematopoietic progenitors obtained from healthy donors. Black arrows in the diagram of RARβ2 promoter/exon 1 region indicate the fragments amplified by the different methylation-sensitive sets of primer pairs (P3 and P4). U and M indicate the unmethylated and methylated forms amplified by methylation-sensitive primer pairs, respectively. The presence of chromosomal translocations is indicated as follows: NN, normal karyotype; c.a., complex aberration. (B) RT-PCR was performed to evaluate the expression levels of RARβ2 mRNA in samples from human AML blasts, normal CD34⁺ hematopoietic progenitors, and an APL-representative patient treated or not “in vitro” for 48 hours with RA (1 μM). GAPDH expression level was used as internal control to evaluate the amount and the integrity of RNA samples.