Loss of Id1 delays the development of MLL-AF9–driven leukemia from FL, and depletion of p21 can rescue Id1-deficiency–induced phenotype. (A) The strategy of FL transplantation model. (B) In the primary transplantation assay, loss of Id1 prolongs the survival time of recipient mice (61 days vs 93 days, n = 15/group, P < .001). Deletion of p21 alone or combined with Id1 deletion does not affect the survival time of recipient mice. (C) In the secondary transplantation assay, the mice in the Id1^−/− group developed leukemia slower compared with the wt group (46 days vs 82 days, n = 15/group, P < .001). Deletion of p21 alone or combined with Id1 deletion does not affect the survival time of recipient mice. (D) The complete blood count analysis showed that, 8 weeks posttransplantation, the average WBC of the wt group is higher than the p21^−/− group, the p21^−/− group is higher than the Id1^−/−p21^−/− group, and the Id1^−/−p21^−/− group is higher than the Id1^−/− group. The average RBC of the wt group is lower than the p21^−/− group, the p21^−/− group is lower than the Id1^−/−p21^−/− group, and the Id1^−/−p21^−/− group is lower than the Id1^−/− group. Platelet (PLT) has no significant differences in these 4 groups. (E) The morphology results showed that there were less leukemia blast cells in the PB, BM, and spleen of the Id1^−/− group mice compared with the wt group. (F) The flow analysis showed that the PB cells of the mice in the Id1^−/− group express less GFP⁺c-Kit⁺ cells compared with the wt, p21^−/−, and Id1^−/−p21^−/− group. (G) The flow analysis of L-GMP (IL-7R-Lin-Sca-1-c-Kit⁺CD34-FcγRII/III⁺ GMP-like leukemic cells) in the BM cells from wt and Id1^−/− MLL-AF9 leukemia mice (left panel). The frequency of L-GMP in the BM cells from Id1^−/− MLL-AF9 leukemia mice is lower than that from wt MLL-AF9 leukemia mice (right panel). (H) The mRNA expression level of Hoxa 9 in wt and Id1^−/− fetal liver HSPCs (marked as wt and Id1^−/−), E14.5 FL HSPCs transduced with MLL-AF9 (marked as MLL-AF9 preleukemia wt and Id1^−/−) and leukemia cells in resulting leukemia (marked as MLL-AF9 leukemia wt and Id1^−/−). (I) The columns represent the numbers of total colonies in each plating of the sorted GFP⁺ wt or Id1^−/− E14.5 FL HSPCs transduced with MLL-AF9 (± standard error of the mean [SEM]; n = 3). CFU-C, colony-forming unit in culture; MSCV, murine stem cell virus.