5 aza dc

Human laryngeal carcinoma cell lines AMC-HN-8, TU212, TU686, and TU177 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were detected and identified as mycoplasma and bacteria free according to ATCC’s instructions during the past 3 months. For drug treatment, the dose and timing of DNA methyltransferase inhibitor 5-Aza-dC and/or histone deacetylase (HDAC) inhibitor TSA were based on similar preliminary studies as well as published studies showing optimal reactivation of gene expression. Cells were seeded the day before the drug treatment. Cells (2 × 10⁵/mL) were treated with 5 μM 5-Aza-dC (Sigma, St Louis, MO, USA) for 72 h, and medium containing 5-Aza-dC was changed every 24 h, or with 0.3 μM TSA (Sigma, St Louis, MO, USA) for 24 h, or with the combination of 5 μM 5-Aza-dC for 48 h followed by 0.3 μM TSA for an additional 24 h. Control cells received no drug treatment. For TGF-β1 treatment, AMC-HN-8 cells were treated with 10 ng/ml of recombinant TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 21 d with TGF-β1 replenishment every 2 days.

UCC cells were seeded in 75 cm² culture flasks at a density of 2 × 10⁵ and incubated at 37ºC in 5% CO2/95% air overnight. Cells were then treated with 5μM of 5-aza-dc (Sigma Chemical, Sigma, USA) for 5 days. Medium with 5-aza-dc was changed daily. Additionally, combination treatment with 5-aza-dc and TSA was performed by adding 5μM of 5-aza-dc daily for 5 days and TSA (300 nmol/L; Sigma) was added to the medium for the final 24 hours. Cells were harvested after the last day of treatment (5-aza-dc only and 5 aza-dc + TSA) for RNA extraction and the analysis of gene expression were performed by Quantitative Reverse Transcriptase-PCR (Q-RT-PCR). PBS (phosphate buffered saline) alone was used as a control to exclude non-specific solvent effects on cells. All experiments were run independently twice.

To evaluate whether spinophilin is directly or indirectly regulated by DNA-hypermethylation or histon-acetylation we treated HCT-116, RKO, Colo320 and Colo201 CRC cells with modifiers of these epigenetic changes. De-methylation was induced with 5-aza-dC (Sigma-Aldrich, St. Lois, US) treatment at different concentrations, which were previously proven to induce maximal de-methylation of the DNA without killing the cells. 100000 cells per well were seeded in 6-well culture dishes and 24 hours later the drug treatment was started. Culture media for HCT-116 and RKO cells contained 1 μM or 15 μM 5-aza-dC. Cells were incubated for 96 hours with 5-aza-dC with the culture media being replaced every 24 hours with fresh media containing 5-aza-dC. RNA was extracted after 96 hours of drug treatment. The experiment was also performed with cells continuously exposed to 40 nM trichostatin A (TSA; Sigma-Aldrich) without 5-aza-dC and as an add-on combinatorial treatment after 72 hours of 15 μM 5-aza-dC to assess the effect of histone acetylation on DNA re-methylation. This chosen concentration has previously been shown to cause hyper-acetylation in HCT-116 cell line [23 (link)].

Human esophageal cancer cell lines (Kyse170, Eca109, TE1, and TE13) were cultured in the RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) at 37 °C and 5% CO₂. Human normal esophageal epithelial cell line HEEpiC was cultured according to the manufacturer’s instructions. When needed, cells (2 × 10⁵/mL) were treated with 5 μM DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC) (Sigma, St Louis, MO, USA) for 72 h with 5-Aza-dC replenishment every 24 h; or with 0.3 μM histone deacetylase inhibitor TSA (Sigma, St Louis, MO, USA) for 24 h; or with the combination of 5 μM 5-Aza-dC for 48 h followed by 0.3 μM TSA for an additional 24 h. Control cells received no drug treatment. The dose and time of 5-Aza-dC and TSA were based on preliminary studies, which showed the optimal gene reactivation^{31 (link)}.

NSCLC cells were treated with different concentrations (1, 2.5, and 5 μmol/L) of the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-Aza-dC, Sigma, St. Louis, MO, USA) or not treated with the 5-Aza-dC for three days. All media were changed daily.