Ring1A/B are crucial for immortalization of MOZ-TIF2–induced AML cells. (A) PCR analysis of genomic Ring1B sequences in Ring1A^−/−;Ring1B^f/f;CreERT2 BM cells treated with 4-OHT or ethanol (vehicle control). CreERT2 BM cells are shown as controls. (B) Experimental scheme for colony formation assay (left) and bar chart of the serial replating experiments (right). The graph shows colony number per 10 000 plated cells in each round of plating in methylcellulose media in the presence or absence of 4-OHT. CreERT2 BM cells served as controls. Error bars represent standard deviation (SD) (n = 3). (C) Representative morphology and May-Grünwald Giemsa staining of MOZ-TIF2 colonies cultured with 4-OHT or ethanol for 7 days. Scale bar, 50 μm. (D) Apoptosis analysis by Annexin V staining. After incubation with 4-OHT or vehicle for 72 hours, MOZ-TIF2 leukemic cells were stained with Annexin V Alexa Fluor 594 and attached to glass slides by cytospin. (E) Kaplan-Meier survival curve analysis of tamoxifen or corn oil–treated mice that received a secondary transplant of MOZ-TIF2−transduced Ring1A^−/−;Ring1B^f/f;CreERT2 BM cells. Two weeks after transplantation, secondary recipients were intraperitoneally administered tamoxifen or corn oil 3 times per week. KO, knockout