Increased C/EBPα-p30 expression after treatment with the immunomodulatory compound, lenalidomide. (A) Western blot analysis of AML patient blasts treated in vitro with 3.0μM lenalidomide followed by hourly collections. The expression of C/EBPα (p30 and p42) was detected by C/EBPα antibody. The signal intensities were assessed and the p30/p42 ratios were calculated (shown below C/EBPα stained blot). Staining with Actin antibody served as an internal loading control. The data shown are representative for 3 patient samples. (B) Quantitative real-time RT-PCR data for miR-181a expression in the same AML blasts shown in panel A. The expression of miR-181a was increased at 12 hours and 24 hours after 3.0μM lenalidomide treatment (black bars) compared with the vehicle control (white bars) for the same time point. Average of triplicate measurements from a single patient sample is shown and error bars denote SEM. Similar results shown in panels A and B were observed in a total of 3 separate AML patient blasts used in the same experiment (not shown). Additional data from the long-term in vitro treatment with lenalidomide are shown in supplemental Figure 3. (C) Quantitative real-time RT-PCR data from 3 bone marrow samples from patients treated with lenalidomide induction therapy (top). Samples were analyzed before treatment (white bars) and 5 days after lenalidomide induction therapy (black bars). Data are shown as average of triplicate measurements and error bars denote SEM. Increased expression of miR-181a was observed on day 5 after lenalidomide induction therapy. Corresponding whole cell lysates for those patients were analyzed using Western blot (bottom). The expression of C/EBPα (p42 and p30) was increased on day 5 after lenalidomide induction therapy. Actin served as an internal loading control. Patients' cytogenetic and clinical characteristics before the therapy are summarized in supplemental Table 1.