5-Aza-2-DC treatment
HepG2 cells at the logarithmic growth stage (1.2×106cells/well) were spread on 6-well plates. After 24 h of culturing, the
cells were treated with 0 μM
(DMSO; 0.1% v/v) or 10 μM
5-Aza-2-DC, and the 5-Aza-2-DC-containing medium was replaced every 24
h. After treatment with 5-Aza-2-DC for 72 h, the medium was discarded
and replaced with a tacrolimus-containing medium (60 μM), and the cells
were cultured for another 48 h. After 48 h, the medium was replaced with
tacrolimus-free DMEM and the cells
were further cultured. Samples of the
medium were taken at 0, 4, 6, and 12 h following its replacement. Next,
the cells were digested with trypsin for 2 min, and then digestion was
terminated by adding more medium. The cells were centrifuged at
300×g for 3 min and washed thrice with PBS. The supernatant of
cell culture and cells were stored at −80 °C for further evaluation.