Decitabine

CRC cell lines (LS174T, T84, LS180, HCT15, HT29, SW620, COLO205, HCT116, COLO320, LoVo, CaCo2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in media with supplements as described in Additional file 1: Table S2 under humidified atmosphere of 5% CO2.
Treatment with DNA methyltransferase inhibitor (DNMTi): 3.5 × 10⁴ COLO205 or SW620 cells were seeded in six-well plates and treated with 0.5% DMSO, 1.25 μM, 2.5 μM, 5 μM, and 10 μM of decitabine (Stock 50 mM in DMSO, Cat.#S1200, Selleckchem, Houston, TX, USA) for 48 h.
Treatment with histone deacetylase inhibitors (HDACi): 3.5 × 10⁴ COLO205 or SW620 cells were seeded in six-well plates and treated with 0.01% DMSO, 50 nM trichostatin A (Stock 5 mM in DMSO, Cat.# T8552, Sigma-Aldrich, St. Louis, MO, USA) and 20 nM LMK-235 (Stock 10 mM in DMSO, Cat.# S7569, Selleckchem) alone or in combination with 2.5 μM, 5 μM, and 10 μM of decitabine for 48 h.
3.5 × 10⁴ HT29, SW620, LS174T, and LoVo cells were seeded in six-well plates and treated with 8 × 10⁻³ DMSO, 5 nM, 10 nM, 20 nM, 40 nM, and 80 nM of LMK-235 for 48 h.

NCI-H1975, NCI-H1650, NCI-HCC827, NCI-H1299, A549 and PC9 cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). They were maintained in RPMI-1640 medium (Gibco-Life Technologies, NY, USA) supplemented with 10% fetal bovine serum (Gibco-Life Technologies), 100 μg/mL streptomycin and 100 U/mL penicillin (Gibco-Life Technologies). Cells were cultured at 37°C with a humid atmosphere of 5% CO₂ and 95% air. Pemetrexed, decitabine, and cycloheximide were obtained from Selleck Chemicals (Houston, TX, USA).

Human HCC cell lines (Bel-7402, HepG2, Huh7, MHCC-97H, MHCC-97L, SMMC-7721, SNU-387, and SNU-449) and immortalized normal liver cell line LO2 were purchased from American Type Culture Collection and Cell Resource Center of Shanghai Institutes. HepG2, Huh7, MHCC-97H, MHCC-97L, and LO2 were cultured in DMEM medium (Gibco) supplemented with 10% FBS (BI). Bel-7402, SMMC-7721, SNU-387, and SNU-449 were cultured in RPMI1640 medium (Gibco) supplemented with 10% FBS (BI). For demethylation treatment, HCC cells were treated with 10μM decitabine (Selleck, S1200) for 3 days and harvested for the following experiments.