Fig. 3: ATM protects the DOT1L protein from ubiquitination-mediated degradation. | Oncogene

Fig. 3: ATM protects the DOT1L protein from ubiquitination-mediated degradation.

From: The DNA damage-independent ATM signalling maintains CBP/DOT1L axis in MLL rearranged acute myeloid leukaemia

Fig. 3

Control (shSCR) and ATM knockdown (shATM) MOLM-13 (A) and MV4-11 (B) 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. C 293T cells were cotransfected with HA-DOT1L and control vector (Flag-Vec) or Flag-ATM. 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. The IP samples of MOLM-13 (D), MV4-11 (E), and 293T (F) cells were detected by western blotting with the indicated antibodies. MOLM-13 (G, H) and MV4-11 (I, J) cells were transduced with lentivirus expressing wild-type (WT) or mutant HA-DOT1L (K358Q, K358R). The cells were incubated with 40 μg/ml CHX for the indicated times. The total lysates of the cells were then subjected to western blotting with the indicated antibodies. The band intensity of HA was normalised to that of ACTB of the same sample, and each time point from three independent experiments was normalised to time 0 for statistical analysis. The difference in slopes between every two groups was compared. ns, not significant. ***P < 0.001. 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 and green fluorescent protein (GFP). The cells were subjected to flow cytometry (see Supplementary Fig. S1C for gating) to analyse the proportion of GFP+ cell. 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 < 0.001. Three independent experiments were performed.

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