Knockdown of ASB2 up-regulates HOXA9 and delays cell differentiation. (A) RT-qPCR confirmation of ASB2 knockdown in NB4 cells after doxycycline treatment for 24 hours. Expression is shown relative to scrambled shRNA-transduced control cells. Error bars indicate SD. (B) Protein level of MLL was detected by Western blotting with whole cell lysate, which shows an increase after doxycycline induced ASB2 knockdown. (C) Expression of HOXA9 was measured by RT-qPCR after doxycycline induced ASB2 knockdown. Expression is shown relative to scrambled shRNA-transduced control cells. Error bars indicate SD. (D-E) NB4 cells were treated with doxycycline for 24 hours to induce ASB2 knockdown and then with ATRA to induce differentiation. (D) Expression of CD11b was determined by flow cytometry at 24 and 48 hours. (E) NBT assays were performed at 24 and 48 hours and the percentage of positive cells is shown. Error bars indicate SD calculated from 3 independent experiments. (F) Control and ASB2 knockdown NB4 cells were grown in liquid culture with or without ATRA treatment. A proliferation advantage was observed from ASB2 knockdown cells in both conditions. Error bars indicate SD.