Fig. 7
From: Disruption of the C/EBPα—miR-182 balance impairs granulocytic differentiation
Enforced expression of miR-182 enhances replating capacity. a Schematic overview of the experimental strategy. Bone marrow cells were isolated from the tibia and femur and ficolled to obtain the mononuclear cells (BM-MNCs). Cells were infected with virus coding for murine miR-182 or scramble control and sorted for GFP + cells two days after infection. Cells were placed in semisolid medium and replated every 7–9 days. b Representative plates of CFU-assay after the fourth round of replating revealed more colonies after enforced miR-182 expression compared to control. c Summary of all replating rounds as a result of 4 independent wells of a 12 well plate. MiR-182 expressing mBM-MNCs showed significantly enhanced replating capacity. Data represent the mean ± SD of four independent wells. (***p < 0.001) P values were calculated using unpaired Student’s t-test. d Exogenous C/EBPα lacking a 3′UTR rescues the ability of miR-182 to enhance replating capacity: Representative plates of CFU-assay after third round of replating revealed more colonies after enforced miR-182 expression (182-ctr). This effect was rescued by additional infection with CEBPA coding lentiviral vectors (182-CEBPA). e Summary of all replating rounds as a result of 4 independent wells of a 12 well plate at the second and third replating round. MiR-182-CEBPA double expressing mBM-MNCs showed significantly reduced replating capacity compared to miR-182-control expression cells. Data represent the mean ± SD of four independent wells. (***p < 0.001) P values were calculated using unpaired Student’s t-test. scr = miR-scramble control vector, ctr = CEBPA empty control vector
