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. 2020 Jan 1;10(4):1758–1776. doi: 10.7150/thno.39013

Figure 2.

Figure 2

DOT1L acetylation confers DOT1L stability to regulate EMT transcription factor expression. (A) HCT116 cells were transfected with Flag-DOT1L(WT), Flag-DOT1L(K358Q) or Flag-DOT1L(K358R), and the Flag immunoprecipitates were incubated with histones extracted from HCT116 cells separately. Western blotting was performed to detect H3K79me1/2/3 levels. (B) Whole cell lysate (WCL) were extracted from HCT116 cells transfected with Flag-DOT1L(WT), Flag-DOT1L(K358Q) or Flag-DOT1L(K358R), and then analyzed by western blotting with the indicated antibodies. (C) HCT116 cells were transfected with Flag-DOT1L(WT), Flag-DOT1L(K358Q) or Flag-DOT1L(K358R) for 36 h, and then incubated with 20 μg/ml cycloheximide (CHX) for the indicated times and analyzed by western blotting. (D) HCT116 cells were transfected with Flag-DOT1L(WT), Flag-DOT1L(K358Q), or Flag-DOT1L(K358R) for 24 h, and subsequently treated with PBS, MG132 (10 μM) or chloroquine (CHQ) (50 μM) for 24 h. WCL were extracted and analyzed by western blotting with the indicated antibodies. (E) HCT116 cells were transfected with Flag-DOT1L(WT), Flag-DOT1L(K358Q) or Flag-DOT1L(K358R) for 24 h, and subsequently treated with PBS or MG132 (10 μM) for 24 h, soluble nucleoplasm proteins (Dt), and chromatin proteins (Chr) were extracted and analyzed by western blotting with the indicated antibodies. (F) DOT1L(K358Q), DOT1L(K358R) or DOT1L(WT) were transfected in HCT116 cells. The lysates were extracted, and DOT1L ubiquitination was detected by co-IP and western blotting with the indicated antibodies. (G) WCL and histones were extracted from HCT116 cells transfected with Flag-DOT1L(WT), Flag-DOT1L(K358Q) or Flag-DOT1L(K358R), and H3K79 methylation was detected by western blotting with the indicated antibodies. (H) ChIP-qPCR showing the level of the indicated proteins recruited to the SNAIL (left) and ZEB1 (right) promoter regions. The data represent the means ± SD (n=3). *p < 0.05. (I) SNAIL and ZEB1 mRNA levels in pcDNA-, DOT1L(WT)-, DOT1L(K358Q)- or DOT1L(K358R)-transfected HCT116 cells were analyzed by RT-qPCR. The data represent the means ± SD (n = 3). *p < 0.05. (J) EMT marker expression was measured by western blotting in HCT116 cells transfected with pcDNA, DOT1L(WT), DOT1L(K358Q) or DOT1L(K358R).