Effect of Aiolos on apoptosis in B cells from CLL patients. (A) Influence of Aiolos knockdown and Aiolos overexpression on antiapoptotic gene expression analyzed by qRT-PCR and normalized by use of the 2^−ΔCt calculation method (Ct indicates cycle threshold) and β-actin as reference gene. Results are expressed as fold down- or up-regulation of transfected cells by pcDNA3.1-Flag-Aio-1 vector or Aiolos siRNA compared with transfected cells by pcDNA3.1-Flag vector or nontargeting negative control siRNA, respectively. The histograms represent the mean ± SD of genes down-regulation (n = 3, left) or up-regulation (n = 2, right). (B) PMA/ionomycin-induced cell death assayed by AV/PI double staining and flow cytometry. B cells from CLL patients transfected with pcDNA-Flag-Aio-1 or pcDNA-Flag vector. Eighteen hours after transfection, cells were stimulated by PMA and ionomycin (10 ng/mL and 1 μg/mL) for 8 hours before apoptosis analysis. Representative dot plots show AV/PI staining on B cells from patient P44. Percentages of viable (AV⁻/PI⁻), apoptotic (AV⁺/PI⁻), secondary apoptotic/necrotic (AV⁺/PI⁺), and necrotic (AV⁻/PI⁺) populations are indicated (left). The histograms represent the mean proportions of AV⁻/PI⁻, AV⁺/PI⁻, AV⁺/PI⁺, and AV⁻/PI⁺ cells obtained with cells from 5 patients (P37-39, P43, P44; right).