miR-181a and miR-181b expression increases after ibrutinib treatment in patients with CLL in vitro. The relative expression of miR-181a (A) and miR-181b (B) was evaluated by RT-qPCR in purified primary CLL cells treated for 72 hours with ibrutinib 10 μM, or DMSO as a control. RNU44 was used as an internal control. The relative expression of miRNAs for each patient was normalized to the DMSO-treated sample. miR-181a expression was assessed in 28 patients with CLL, with 21 analyzed in experimental duplicates and 7 (CLL45B, CLL176, CLL192, CLL197, CLL198, CLL210, and CLL211) analyzed in a single experiment. Similarly, miR-181b expression was evaluated in 27 patients with CLL, with 24 analyzed in duplicates and 3 (CLL161, CLL176, and CLL198) in a single experiment. The Wilcoxon matched-pairs test was used for statistical significance. (C) Cell viability of purified primary CLL cells was assessed after treatment with increasing doses of ibrutinib or DMSO (0, 10, and 100 μM) for 72 hours using the MTS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium)) assay. Statistical significance was determined by 1-way analysis of variance (ANOVA). (D) Cell viability of primary CLL cells transfected with either pTween_181b, pTween_181a, or pTween_CTRL vectors was evaluated after a 72-hour treatment with increasing concentrations of ibrutinib or DMSO (0, 10, and 100 μM). Cell viability was determined using the MTS assay. Statistical significance was assessed by 1-way ANOVA. DMSO, dimethyl sulfoxide.