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-NucleofectorTM 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.