In vitro analysis of cultured bone marrow cells. (A) Shown are images from the cultured bone marrow preparations illustrating the increased proliferation of hTg^WT cells compared with hTg^Y605X cells (for comparison, see also Table 1). Images were captured with an Olympus IX71 inverted microscope equipped with a 40 ×/0.95 NA objective (Olympus, Tokyo, Japan). Images were photographed at 400 × original magnification with an Olympus DP70 CCD camera (Olympus) and acquired through Lumina Vision software (Mitani, Fukui, Japan). Cells were stained for acetylcholinesterase using 0.1 mol/L PBS containing 0.05% acetylthiocholine iodide, 0.1 mol/L sodium citrate, 30 mmol/L copper sulfate, and 5 mmol/L potasium ferricyanide (pH 6.0). (B) TUNEL assay on cultured bone marrow cells. Bone marrow cells were harvested and cultured for 5 days in the presence of TPO, and TUNEL assays were performed. Flow cytometry settings were used to gate and provide data for cells with greater than 4n ploidy. (C) Western blot from the same cultures depicted in the center panel was blotted for GP Ibα antigen, a marker of late-stage megakaryocytopoiesis. Ten micrograms protein (BCA assay) was applied to each lane, electrophoresed, transferred to nitrocellulose, and reacted with an anti–GP Ibα antibody. Shown is the resultant autoradiograph. Samples from hTg^Y605X show an increase in the amount of GP Ibα antigen compared with hTg^WT, consistent with an increase in high-ploidy cells seen after a 5-day culture in the presence of TPO. However, as seen in Figures 1 and 2, gene expression levels for both transgenes were similar, but coincident with an increase in the percentage of high-ploidy megakaryocytes was an increase in GP Ibα antigen. The blot was subsequently reprobed with a pool of antibodies to confirm a similar protein load and is shown below for comparison.