5 aza 2 deoxycitidine 5 aza dc

Selected stable and unstable clones generated using both promoters were seeded into 24-well plates at cell density of 3 × 10⁵ cells/mL and cultured for 24 h in selection free DMEM. Media was changed to fresh DMEM containing either 4 μM 5-Aza-2′-deoxycitidine (5-Aza-dC, Sigma-Aldrich), a DNMT inhibitor, or 2 mM sodium butyrate (NaBu, Sigma-Aldrich), a HDAC inhibitor, or without inhibitors as a control. Cells were then further cultured in the DMEM supplemented with inhibitors for 24 h before being harvested for FACS analysis to obtain the MFI of the clones after treatment.

The human pancreatic duct epithelial cells (HPDE) and pancreatic cancer cell lines (PANC-1, BxPC-3, HPAF-II and SW1990) were obtained from the Cell Bank, Chinese Academy of Sciences (Shanghai, China) and propagated in our laboratory by culturing in complete growth medium as recommended by the supplier. All cell lines were genotyped for identity by Cell Bank, Chinese Academy of Sciences and tested for ruling out mycoplasma contamination. Cells were incubated at 37 °C in atmospheric conditions of 20% O₂ and 5% CO₂, treated with 4 μM 5-aza-2′-deoxycitidine (5-Aza-dC) (Sigma, St. Louis, MO, USA) for 3 days, before harvesting for assays for methylation and mRNA expression.