2.6 Transient transfection

Cells were respectively cultivated in 48-well plates overnight. Plasmids were transfected into HEK293 cells by lipofectamine 3000 (Invitrogen), and siRNAs were transfected into Raji, MV4-11, and Jurkat cells by SG Cell Line 4D-Nucleofector^TM X Kit (Lonza). The experiments were performed in triplicate for each trial.

2.7 Dual-luciferase reporter assays

Putative GREs in the human DOT1L promoter region (-2,800 bp to +200 bp) were predicted using NUBIScan, an online Algorithms. Then theDOT1L gene promoter region (-1,800 bp to +200 bp) which including GREs was amplified with PCR and cloned into pGL3-basic vector (Promega), and the recombinant reporter plasmids was defined as pGL3-DOT1L. For reporter assays, the pGL3-DOT1L or pGL3-basic plasmids were separately co-transfected with pRL-TK (Promega) expressing Renilla luciferase into HEK-293 cells using lipofectamine 3000. After incubating of 18 h, Dex was added to the medium with or without RU486 for 6 h. Then, cells were collected and measured using Dual-Luciferase Assay System (Promega). To adjust the differences in the aboved experiment, pRL-TK was co-transfected as an internal control. Each assay was done in triplicate.

2.8 Statistical analysis

The data were exhibited as means ± standard deviation. Student’s t-test and One-way ANOVA was applied to compare between two groups or multiple groups respectively to calculate the statistical significance (p value) for all data using Prism 6.0 (GraphPad). In all cases, ‘ns’ indicated no significance, while P < 0.05 (‘*’) was regarded as statistically significant.