SU6656 induces polyploidization. (A) UT-7/TPO cells were grown in the presence of 10 ng/mL rhTPO + 0.1% DMSO or 10 ng/mL rhTPO + 2.5 μM SU6656 for 48 hours. Cell were partially permeabilized, incubated with RNase and propidium iodide (10 ng/mL), and analyzed by flow cytometry to detect changes in nuclear ploidy under each condition: T = 0, 12, 24, and 48 hours. (B) HEL cells (upper panels) and K562 cells (lower panels) were each cultured for 72 hours under normal growth conditions (+ 0.1% DMSO) or in the presence of 2.5 μM SU6656 (+ 0.1% DMSO). Nuclear ploidy was determined by flow cytometry. Bars mark the DNA contents of 2N, 4N, 8N, 16N, and 32N. (C) Cells were cultured for 48 hours with 10 ng/mL rhTPO in the absence or presence of 2.5 μM SU6656. Morphologic changes were examined by staining cytocentrifugation preparations with Wright-Giemsa (magnification × 100). (D) Cytoplasmic protrusions are seen in the presence of SU6656 as show by Wright-Giemsastained UT-7/TPO cells at higher magnification (× 400) after 72 hours. (E) UT-7/TPO cells were cultured from an initial density of 1 × 105/mL in media containing rhTPO ± SU6656 (2.5 μM). Each day, the cell number was counted using a hemocytometer and methylene blue to exclude nonviable cells. (F) Flow cytometric analysis of indirect immunofluorescence staining of SU6656-treated and untreated cells with anti-CD41-APC, or CD61-APC, and an isotype-matched irrelevant control APC antibody. The percentage of CD41/CD61 cells is an average of 4 independent experiments. Error bars indicate standard deviation. Mean values are significantly different from untreated cells (CD41, P < .05; CD61, P < .01).
