MPLS204P does not present proliferative advantage in bulky cultures. (A-B) Cell proliferation induced by TPO of MPLWT and MPLS204P UT7 cells. Cells were cultured in the presence of various concentrations of TPO (A) or GM-CSF (B) for 72 hours. Viable cells were quantified by WST-1 proliferation assay. Dose-response curves are means ± SEM (n = 3 in triplicate). (C-D) Apoptosis analysis in MPLWT and MPLS204P UT7 cells. (C) Cells were cultured for 2 days with various concentration of TPO (0.01, 0.05, and 10 ng/mL) and the percentage of apoptotic cells (Annexin V positive) was analyzed by flow cytometry using the Annexin V assay. (D) Cells were cultured for 24 hours with TPO (10 ng/mL) followed by overnight deprivation. The percentage of apoptotic cells (Annexin V-positive) was analyzed at different times after TPO removal (1, 2, 3, and 4 days). (E) Cell-cycle analysis of MPLWT and MPLS204P UT7 cells. Cells were cultured for 2 days with various concentrations of TPO (0.01, 0.05, and 10 ng/mL) and the percentage of cells in G1, S, and G2 phases were determined after propidium iodide labeling, by flow cytometry analysis. SEM, standard error of the mean.