Analysis of Aiolos and Ikaros expression in CLL. (A) Quantification of total Aiolos transcripts in B cells from 32 CLL patients (P1-P18 and P20-P33) and 28 HDs obtained by RT-qPCR of B-cell RNA and normalized to the Abl gene. Absolute quantification of Aiolos and Abl was performed with the use of known quantities of pcDNA3.1-hAio1 construct and Daudi total RNA, respectively. Respective data are summarized and presented as the mean ± SEM. (B) Comparison of Aiolos levels in CLL subsets defined by V_H gene mutation status, ZAP-70, and CD38 expression. Respective data are summarized and presented as the mean ± SEM. Differences between groups were tested using unpaired t test (Prism5.0c software; ns indicates non significant; *P < .05; **P < .01; ***P < .001). (C) Aiolos protein levels in PBMCs from 7 CLL patients and 2 HDs were detected by Western blot with a specific anti-Aiolos antibody. Actin was used as internal loading control (top). The histogram shows the densitometric analysis of 2 independent Western blot experiments with same protein samples. The error bars represent standard deviation (bottom). (D) Expression of Ikaros transcripts in PBMC from HD and CLL patients. Representative results of Ikaros isoforms expression in 2 HD and 5 CLL patients (P2, P25, P31, P32, and P34) after one-step RT-PCR amplification of PBMC RNA. β-actin was used as internal control. (E) Quantitative analysis of total Ikaros mRNA levels in B cells from 13 HD and 14 CLL patients (P17, P18, P20-P23, P25, P26, and P28-P33). Data are expressed as normalized expression by use of the 2^−ΔCt calculation method (Ct indicates cycle threshold) and β-Actin as reference gene. n.s. indicates nonsignificant; *P < .05; **P < .01; ***P < .001.