(A, D) The methylation status of the CpG island and CpG shore regions of the Shh gene were analyzed by bisulfite sequencing in wild-type bladder tissues, BBN-induced bladder tumors with or without 5’-azacitidine treatment (A), and tumor organoids with or without 5’-azacitidine treatment (D). BBN-induced mouse tumors were orthotopically transplanted and, 1 week after transplantation, the resulting animals were treated with 5’-azacitidine (1 mg per kg mouse body weight) every other day for 2 weeks before methylation analysis. The gene discussed is SHH; the disease is neoplasm.