Fig. 4: CBP-mediated DOT1L-K358 acetylation confers DOT1L stability in MLLr-AML.

A, B The cells were transduced with lentivirus expressing control shRNA (shSCR) or shRNA targeting CBP (shATM) along with GFP. The cells were subjected to flow cytometry (same gating strategy as in Supplementary Fig. S1C) to analyse the proportion of GFP⁺ cells among the MOLM-13 (A) and MV4-11 (B) cells. The analysis was performed every two days during the culture. Three replicates for each time point for different cells were used for linear regression analysis. The difference in slopes between every two groups was compared. ***P < 0.001. Three independent experiments were performed. The total lysates of the control (shSCR) and CBP knockdown (shCBP) MOLM-13 (C) and MV4-11 (D) cells were subjected to western blotting with the indicated antibodies. Control (shSCR) and CBP knockdown (shCBP) MOLM-13 (E) and MV4-11 (F) cells were incubated with 25 μM MG132 for 4 h. The cell lysates were subjected to immunoprecipitation (IP) with DOT1L antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. G HA-DOT1L with or without Flag-CBP were cotransfected into 293T cells. The cell lysates were then subjected to western blotting with the indicated antibodies. H 293T cells were cotransfected with HA-DOT1L and a control vector (Flag-Vec) or Flag-CBP. At 36 h posttransfection, the cells were treated with 25 μM MG132 for 20 h. The cell lysates were subjected to immunoprecipitation (IP) with HA antibodies. The input and IP samples were then detected by western blotting with the indicated antibodies. I, J MOLM-13 (G) and MV4-11 (H) cell lysates were subjected to IP with CBP, DOT1L, or control IgG. The input and IP samples were then detected by western blotting with the indicated antibodies. MOLM-13 (K) and MV4-11 (L) cells were transduced with lentivirus expressing HA-DOT1L (K358Q) or not (vector). The cells were then transduced with lentivirus expressing shRNA along with green fluorescent protein (GFP). The cells were subjected to flow cytometry (See Supplementary Fig. S1C for gating) to analyse the proportion of GFP⁺ cells. The analysis was performed every two days during the culture. Three replicates for each time point for different cells were used for linear regression analysis. The difference in slopes between every two groups was compared. ns, not significant. *P < 0.05, ***P < 0.001. Three independent experiments were performed.