THPO/MPL signaling regulates cell cycle reentry and prevents AE cell differentiation. (A) CD34⁺ AE/pBabe or AE/Bcl-2 cells were incubated with (+) or without (−) THPO for 3 days, followed by stained with annexin V and analyzed by flow cytometry. (B) CD34⁺ AE/pBabe or AE/Bcl-2 cells were cultured with (+) or without (−) THPO. Cell counts were performed biweekly to obtain growth curves of the cells in each condition. (C-D) Cell cycle exit analysis. AE cells washed with PBS 3 times were incubated with or without THPO (in the presence of KF36) in media containing 10μM BrdU for 24 hours. BrdU incorporation and Ki-67 marking were determined by flow cytometry (*P < .05). (E) AE/pBabe and AE/Bcl-2 cells cultured with or without THPO (in the presence of KF36) for 14 days were labeled with anti-CD34 antibodies for flow cytometry analysis.