Effect of RUNX1 knockdown on in vitro megakaryopoiesis. CD34⁺ cells from control individuals were transduced with a lentivirus encoding either scramble shRNA (control) or RUNX1_1 shRNA or RUNX1_2 shRNA. Transduced CD34⁺ cells were grown in liquid medium in presence of TPO, IL3, IL6, SCF, and FLT3-L. (A) Flow cytometry analysis was performed after 10 days of culture. A representative analysis of 3 repeated experiments is shown. (B-C) CD41⁺CD42⁺GFP⁺ cells were sorted at day 10 of culture and seeded at 2 × 10³ cells/well in 96-well plates. The percentage of proplatelet forming MKs was estimated at day 13. (B) Representative microscopic images representing control and shRUNX1_2 transduced proplatelet forming MKs. (C) The percentage of proplatelet forming MKs was estimated by counting MKs exhibiting 1 or more cytoplasmic processes with areas of constriction. A total of 500 cells per well was counted. The histograms show 1 representative experiment of 3, each in triplicate. Error bars represent ± SD of triplicate (n = 3 for both shRUNX1_1 and shRUNX1_2, P < .05).