2. Materials and Methods
2.1 Reagents
Dex, RU486, and actinomycin D (Act D) were obtained from Sigma-Aldrich
(St Louis, USA). siRNAs for DOT1L and negative control were synthesized
by Invitrogen, the sequences for DOT1L
siRNA
included:
siRNA-1,
5’-CGCGAGUUCAGGAAGUGGAUGAAAU-3’; siRNA-2,
5’-CGAUAAACAUCACGAUGCUGCUCAU-3’; and siRNA-3,
5’-CGCUGCCGGUCUACGAUAAACAUCA-3’. Dual-luciferase reporter system was
bought from Promega (Madison, USA). Primary antibody against H3K79me2
and H3 were purchased from Cell Signaling (Danvers, MA) and Thermo
Scientific (Rockford, USA) respectively, and the secondary antibodies
were bought from Zhongshan Biotechnology.
2.2 Cell culture
Human cell lines Raji, Daudi, Namalwa, JeKo-1, THP-1, Jurkat, MV4-11,
and HEK-293 were acquired from the American Type Culture Collection
(ATCC) and then cultivated in a humidified incubator set to 37℃,5%
CO2. Raji, Daudi, Namalwa, JeKo-1, THP-1, and Jurkat
cells were grown in RPMI-1640 medium (Gibco) with
10%
Fetal Bovine Serum (FBS). MV4-11 and HEK-293 cells were grown in IMDM
and DMEM medium with 10% FBS respectively.
2.3 Western blot analysis
Histones of each human cell line were prepared by the EpiQuik™ Total
Histone Extraction Kit, and the protein concentrations were calculated
using BCA protein assay kit. Then 3 µg histones were loaded on 15%
SDS-PAGE and following transferred to PVDF membranes (Millipore). After
subsequent blocking with 5% fat-free dry milk for 2 h, the membranes
were probed overnight at 4℃ with antibodies against H3K79me2 and H3,
followed by the relevant horseradish peroxidase conjugated secondary
antibodies (Zhongshan Biotechnology, China). The Supersignal West Dura
Extended Duration Substrate was utilized for signal detection.
2.4 Cell viability assay
Cells were cultivated overnight in 96-well plates at a density of 2 ×
103 cells/well, then conducted with Dex or DMSO in the
presence or absence of RU486. After that, OD value at 450 nm was
obtained by Cell Counting Kit-8 (CCK-8) (Dojindo laboratories, Japan).
The results were normalized against the OD450 values of
the control. The assay was conducted in triplicate.
2.5 Quantitative real-time PCR
Total RNA from cells was extracted by Trizol reagent (Invitrogen, USA),
and 1,000 ng of total RNA was reverse transcribed into cDNA utilizing
PrimeScript™ RT Master Mix (Takara Dalian, China). Quantitative
real-time PCR (qPCR) was conducted in triplicate with SYBR® Select
Master Mix (Applied Biosystems, USA), with β-actin served as the
control. The qPCR experiment was performed using the ABI Prism 7500
detection system. Finally, we used 2 −ΔΔCt method to
calculate the relative mRNA levels of the target genes. The primers were
provided in Table 1.