Lenalidomide treatment in vivo induces miR-181a-mediated inhibition of xenograft AML tumor growth. (A) THP-1 cells were transiently transfected with empty expression vector (control), or construct expressing the ectopic miR-181a and injected subcutaneously into NOD/SCID mice (5 mice per construct). Six weeks later the tumors were excised (left) and their sizes were determined (right). (B-E) THP-1 cells were xenografted subcutaneously to NOD/SCID mice (1 tumor per mouse). Four weeks later, tumors were directly injected with lenalidomide (lenalid.; 50 mg/kg; n = 5) or vehicle control (n = 3), twice a week for 2 weeks. Six weeks after transplantation mice were killed and tumors excised. (B) Xenograft tumors assessed at the onset (white bars) and the end (black bars) of the treatment. Measurements were plotted as the relative percentages of the tumors at the beginning of the treatment. (C) Dissected tumors after the lenalidomide treatment (lenalid.; on the right) or vehicle control (on the left). (D) Quantitative real-time RT-PCR assessment of miR-181a expression for the xenografts after treatment with either lenalidomide (lenalid.; black bar) or vehicle (white bar). (E) The relative expression of C/EBPα-p42 and C/EBPα-p30 in lenalidomide (lenalid.) or vehicle-treated xenograft tumors was evaluated by Western blot of nuclear extracts prepared from the tumors. Blot was stained with C/EBPα antibody and staining with Ku70 served as a loading control. Both lanes were from the same blot and same exposure. Vertical line has been inserted to indicate repositioned gel lanes.