Fig. 6

Exogenous C/EBPα abrogates miR-182 mediated effects. a MiR-182 levels in 32D cells transduced with either miR-182 or scramble lentivirus. MiR-182 expression was measured by qPCR. b MiR-182 expressing 32D cells showed increased cell numbers under G-CSF conditions (black line) compared to control scramble transduced cells (gray line). 1 × 10⁵ cells were initially seeded and cultured in medium without IL-3 and supplemented with 20 ng/ml G-CSF. Data represent the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01) P values were calculated using unpaired Student’s t-test. c Morphological analyses by cytospins followed by Wright-Giemsa-staining (top panel). MiR-182 expressing 32D cells exhibited less morphological signs of mature granulocytes and remained in an immature stage after treatment with G-CSF for 7 days. Representative plate from a CFU-assay of 32D-scramble or miR-182 expressing cells after 7 days under G-CSF conditions (bottom panel). Scale bar 5 µM. d Summary of 4 independent CFU-assays demonstrated enhanced colony forming unit ability of miR-182 expressing 32D cells after 7 days under G-CSF conditions. Data represent the mean ± SD. (**p < 0.01) P-values were calculated using unpaired Student’s t-test. e and f Stable overexpression of miR-182 led to a reduced percentage of Mac-1 positive 32D cells under both IL-3 and G-CSF conditions. e Representative flow cytometry analysis for myeloid marker Mac-1 in miR-182 or scramble expressing 32D cells under IL-3 conditions (left panel) or after treatment with 20 ng/ml G-CSF for 7 days (right panel) and f summary of all experimental repetitions. Y-axis indicate the percentage of Mac-1 positive cells. Data represent the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001) P-values were calculated using unpaired Student’s t-test. g Western blot analysis of 32D cells stably expressing either scramble or miR-182 sequence revealed reduced C/EBPα protein levels under IL-3 conditions or after treatment for 7 days with 20 ng/ml G-CSF. h Transduction of 32D cells with exogenous murine CEBPA enhances the detectable amount of C/EBPα protein. Analysis was performed using western blot. i Exogenous C/EBPα lacking a natural 3′UTR can rescue the miR-182 induced phenotype in 32D cells. Representative flow cytometry analysis for Mac-1 in either parental 32D cells or 32D cells stably expressing miR-182 transduced with control or mCEBPA. Analysis was made 10 days after transduction