PEAR1 knockdown via lentiviral vector transduction. (A) PEAR1 expression at day 10 after transduction of human CD34+ with control (LV_miR_DsRed) or LV_miR_PEAR1_1461 vector analyzed via qRT-PCR (left) and western blotting with PEAR1-EC Ab using HDAC-1 as loading control (middle). Relative suppression of the GAPDH-normalized PEAR1 protein expression at day 11 following transduction of both LV_miR_PEAR1 vectors vs control (right). (B) Relative cell proliferation at day 10 after treatment with both LV_miR_PEAR1 vectors vs control. (C) Numbers of CFU-MK and CFU-E colonies at days 6 and 10 after transduction of human CD34+ with control (black) or LV_miR_PEAR1_1461 (gray) vectors. (D) Coexpression of CD41 and CD42b during CD34+ cell differentiation analyzed by flow cytometry at day 12 after transduction of human CD34+ with control or LV_miR_PEAR1_1461 vectors, as indicated. (E) Polyploidy profiles (left) on day 10, representative of 3 experiments, after anti-CD41 labeling and DNA staining with propidium iodide (PI), after transduction of human CD34+ with control or LV_miR_PEAR1_1461 (n = 3); Ploidy histogram for control and LV_miR_PEAR1_1461 treated CD34+ cells on day 12 (n = 3). 1 A.U., 1 copy for 105 copies of housekeeping gene.
