The methyltransferase inhibitor Aza-dC (Sigma, USA) was used to reverse methylation. The cells were treated with Aza-dC at final concentrations of 0.1, 0.5, and/or 1.0 μM and co-cultured for 3 days prior to the analysis. Untreated controls were prepared in parallel by addition of an equal volume of DMSO.
Aza dc
Manufactured by Merck Group
Sourced in United States
Aza-dC is a chemical compound used in research laboratories. It functions as a DNA methyltransferase inhibitor, which can be utilized for studying epigenetic mechanisms in various biological systems.
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Lab products found in correlation
5 protocols using aza dc
1
Epigenetic Regulation via Aza-dC
2
Reagents and Antibodies Utilized
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AzadC was purchased from Sigma-Aldrich (St. Louis, MO); CpG methyltransferase (M.Sssl) was from New England Biolabs (Ipswich, MA); Decade markers (= 10-nt ladder) for small RNA Northern and yeast tRNA were from Ambion (Carlsbad, CA); and protein and DNA size markers were from GenDepot (Barker, TX). The source of antibodies was described in [1 (link), 2 (link)].
3
Analyzing SSTR2 Expression in Cells
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Cells were seeded at a density of 1 × 106 per 100-mm dish, cultured for 24 or 72 h, and treated with 1 μM 5 of -aza-dC (Sigma-Aldrich, St. Louis, MO, USA) as a demethylating agent, and then with 0.5 μM of TSA (Sigma-Aldrich) as an inhibitor of histone deacetylase. After 2–5 d, cells were washed with PBS, and total RNA was isolated using RNeasy Kit (QIAGEN). The RT-qPCR analysis of SSTR2 expression was performed as described in Section 2.7 . Independent triplicate experiments were performed.
4
Characterization of UROtsa Cell Line
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We used the UROtsa cell line derived from the ureter epithelium of a 12-year-old female donor, immortalized with SV40 large T-antigen [18 (link)]. It is a non-tumorigenic cell line that displays the phenotypic and morphologic characteristics resembling primary transitional epithelial cells of the bladder [19 (link)]. UROtsa cells were cultured in 25 cm2 tissue culture flasks in Dulbeco’s modified Eagle’s medium supplemented with 5% vol/vol fetal bovine serum [20 (link),21 (link)]. Cells were maintained at 37 °C in humidified incubators with 5% CO2/95% atmospheric air. Cells were fed fresh growth medium every three days. At confluence, cells were subcultured at a 1:4 ratio using trypsin-EDTA (0.05%, 0.02%). For experimentation, cells were grown in 6-well plates containing 2 mL growth media per well (35 mm in diameter). Treatment of cultures with Cd [26 (link)], Zn, BSO [27 (link)], and aza-dC [28 ] were undertaken when cultures reached 80% confluence. Cd, Zn, BSO, and aza-dC were of highest purity grade (Sigma, St. Louis, MO, USA). For an initial dose-response analysis, five cell cultures were used for each Cd concentration. Expression of ZIP and ZnT genes from the initial experiment agreed with microarray data using the Affymetrix 133 Plus 2.0 [29 (link)]. Thus, in later experiments, at least two different batches of cell cultures were used per treatment.
5
Culturing Human Teratocarcinoma and ES Cells
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Human teratocarcinoma stem cells N2102Ep and TERA1 (Andrews et al, 1980 (link)) and NTERA2 (Andrews et al, 1984 (link)) were grown using DMEM (high glucose, no pyruvate) (PAA) supplemented with 10% fetal calf serum (FCS; Hyclone, Rockford, IL, USA). N2102Ep and TERA1 were passaged by 0.25% trypsin/EDTA at day 5 of culture as previously described (Andrews et al, 1980 (link)), whereas NTERA2 were passaged at 1 : 3 ratio by using glass beads. The human ES cell line H7 (Thomson et al, 1998 (link)) was grown in knockout-DMEM supplemented with 20% knockout-serum replacement, 1X non-essential amino acids, 1 mM glutamine, 0.1 mM beta-mercaptoethanol and 4 ng ml−1 bFGF (Invitrogen, Grand Island, NY, USA) seeded on 6 × 103 cells cm−2 mitomycin C-treated MF-1 mouse embryonic fibroblasts, and placed at 37 °C under a humidified atmosphere of 5% CO2 incubator. Cells were passaged every 5–7 days using collagenase type IV (Invitrogen) and scraped with glass beads (Sigma, St Louis, MO, USA). For Aza-dC treatment, cells were grown in medium supplemented with 10 nM Aza-dC (Sigma); cultures were maintained for 7 days during which the culture medium was changed every other day.
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