ID1 knockout in BMSCs suppresses AML cell proliferation in vitro and in vivo. (A)The western blot analysis of ID1 expression in HS-5 cells with ID1 knockout and in control cells. (B-F) MTT assays show that ID1 knockout in HS-5 cells significantly inhibits the proliferation of cocultured Kasumi-1, SKNO-1, THP-1, and MOLM-13 cells (n = 3). (G-K) Cell cycle analysis on Kasumi-1, SKNO-1, THP-1, and MOLM-13 cells cocultured with ID1^+/+ or ID1^−/− HS-5 cells for 4 days (n = 3). (L) The strategy of AE9a- and MLL-AF9–expressing cell line transplantation. (M) Id1^−/− recipients showed a significantly longer survival time than Id1^+/+ recipients after undergoing transplantation with AE9a (n = 9; median survival, 24 vs 38 days) or MLL-AF9 cell line (n = 10; median survival, 25.5 vs 33.5 days). (N) The in vivo bioluminescence imaging and quantification analysis shows that the Id1^−/− BMM impairs leukemia progression in recipients that received transplantation with AE9a or MLL-AF9 cell lines that are luciferase positive (n ≥ 4). ∗P < .05; ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. Ctrl, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI, propidium iodide; sg, single guide.