Azacitidine

2 × 10⁵ cells of MIA PaCa-2, PANC-1 and hTERT-HPNE were plated in 6-well plates. On the next day, cells were washed and treated with either azacitidine (Sigma-Aldrich) or decitabine (Sigma-Aldrich) for the following 96 h. During the treatment period, cells were washed daily and fresh medium containing the appropriate concentration of the demethylating agent was used. In azacitidine (Sigma-Aldrich) and decitabine (Sigma-Aldrich) treatments, PANC-1 were treated with a final concentration of 5 μM and MIA PaCa-2 and hTERT-HPNE were treated with a final concentration of 1 μM. Vehicle-treated cells (water/acetic acid 1:1 v/v) were used as control for both azacitidine (Sigma-Aldrich) and decitabine (Sigma-Aldrich) treatments. Technical triplicates were used in each experiment.

Female Sprague–Dawley rats weighing between 200 and 230 g were purchased from the National Laboratory Animal Center. Urinary bladder tumors were induced by administration of 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN, Sunshine Chemical), dissolved in drinking water, for 20 weeks, as described previously [22 (link)]. The therapeutic effects of intravesical azacitidine (CAS no.320-67-2, Sigma-Aldrich, St. Louis,, MO, USA) on bladder tumorigenicity in BBN-induced rats were determined by IVI of azacitidine at 1, 5, 9, 13, and 17 weeks. The IVI protocol was as follows: all rats were anesthetized by intraperitoneal administration of Rompun (6 mg/kg) and Zoletil (30 mg/kg). A sterile polyethylene catheter (PE-50) was then inserted into the bladder through the urethra, and all urine was aspirated. The rats in the BBN-induced group underwent IVI with 0.2 mL azacitidine (0, 10, or 50 μM; n = 5, n = 6, n = 6, respectively; control without BBN, n = 3) under 1 h of continuous sedation. The non-BBN-induced group of rats underwent IVI with 0.2 mL PBS under 1 h of continuous sedation. The rats were euthanized by asphyxiation with CO₂ 20 weeks after BBN induction. Dissected rat bladders were photographed and used for further analysis.

Ponatinib, cladribine, cytarabine, selinexor, JQ1 and venetoclax were purchased from Selleck Chemicals (Houston, TX, USA), dasatinib from ChemieTek (Indianapolis, IN, USA), rapamycin from Calbiochem (San Diego, CA), hydroxyurea (HU) and azacitidine from Sigma-Aldrich (St. Luis, MO, USA). Stock solutions of most drugs were prepared by dissolving in dimethylsulfoxide (DMSO; Sigma-Aldrich); azacitidine was dissolved in 50% acetic acid, and HU in distilled water. RPMI 1640 medium and antibiotics (penicillin, streptomycin) were purchased from Lonza (Basel, Switzerland), amphotericin B from PAN‐Biotech (Aidenbach, Germany), fetal calf serum (FCS) from Gibco Life Technologies (Carlsbad, California, USA), and ³H-thymidine from Perkin Elmer (Waltham, MA, USA).