CD49d expression is correlated with DNA methylation levels. (A) DNA methylation was studied within the CpG island of the ITGA4 gene (reported in supplemental Figure 5). The upper graph reports the percentage of methylation calculated as the number of methylated CpG over the total number of CpGs. The lower graph reports the percentage of sequences with at least 2 methylated CpGs. At least 10 clones for each of 10 CD49d⁺/trisomy 12 (black histograms) and 14 CD49d^–/no trisomy 12 (gray histograms) CLL cases were analyzed. (B) Correlation between the percentage of methylation and CD49d protein expression, as detected by flow cytometry (upper panel) and mRNA as detected by qRT-PCR (lower panel). ρ and P values refer to the Spearman’s rank correlation test. (C-E) Primary CLL cells from 7 cases were cultured for 96 hours using CpG-ODN/interleukin-2 in the presence or not of 5 μmol/L DAC and analyzed by flow cytometry for the surface expression of CD49d. (C) Dot plots of CD19-APC vs BrdU-FITC (upper panels) and CD49d-PE vs BrdU-FITC (lower panels) in untreated and DAC-treated CLL cells from a representative case (case 13). The proliferative/BrdU⁺ and nonproliferative/BrdU^– fractions are noted in red and gray, respectively. (D) Proliferation levels (mean percent ± SEM) of untreated (gray bar) and DAC-treated (black bar) CLL cells after 96 hours in culture with CpG-ODN/interleukin-2. (E) CD49d MFI (mean expression ± SEM) by proliferative/BrdU⁺ (red line) and nonproliferative/BrdU^– (gray line) fractions in untreated and DAC-treated CLL cells; P values refer to the Student t test.