An Aiolos overexpression in CLL patients who seem independent of Aiolos promoter epigenetic modifications. (A) Total amounts of Aiolos transcripts in each subgroup of patients and healthy subjects of the set 2 (Table 2) obtained by reverse-transcribed quantitative polymerase chain reaction of B-cell RNA by the primer pair/probe couple 1 and normalized to the Abelson gene (Abl, Applied Biosystems, Hs00245445_m1). Absolute quantification of Aiolos and Abelson was done using known quantities of pcDNA3.1-hAio1 construct and Daudi total RNA, respectively. Differences between groups were tested using one-way ANOVA and Tukey multiple comparison tests (Prism4.0c software; ns indicates nonsignificant, ie, P > .05). (B) Western blot analysis of Aiolos and Bcl-2 expression in a healthy donor (H) and in the CLL patients P1, P3, P22, and P24. 14-3-3 protein is used as internal control. Molecular weights of the proteins are shown. (C) Schematic representation of the position of the primer pairs used to analyze chromatin remodeling and levels of trimethylated H3K4 (Abcam, Cambridge, MA; AB8580, 1/250) and acetylated H3K9 (Upstate Biotechnology, Charlottesville, VA; 07 352, 1/100) obtained in the CLL patients P1, P3, P22, and P24, and in one healthy donor H. The y-axis corresponds to the percentage of immunoprecipitated chromatin. The x-axis corresponds to the position in the promoter of the chromatin modification. The corresponding Aiolos transcript amounts are shown. Primers sequences were as follows: A1 forward 5′-TGGTCACTTCCCCTTTCCTCT-3′, A1 reverse 5′-TCCCAACACCCTCCCTACTG-3′; A2 forward 5′-CAGTTCCCAGAGGGAAAACAAA-3′, A2 reverse, 5′-TTGGTCCAAGTTTTCAGACAGTTG-3′; A3 forward 5′-GACAAAAAGTTCCAGATCTTCCTCA-3′, A3 reverse 5′-GTCAGCCTTGCTTTCTTGGc-3′; A4 forward 5′-GAGAGGCCGAGTAGCCACAG-3′, A4 reverse 5′-TTGCACAGGTTAAGTTTCTCAAAGA-3′; A5 forward 5′-TTGGCATTTGCAGTTCCCTT-3′, A5 reverse 5′-AACCTGATTTTTTGCGCTGG-3′; A6 forward 5′-TGGGCTGTTGTATACTATGGGAAA-3′, A6 reverse 5′-CGGCTAGGAAAAATAGTGTTGGA-3′.