Female Sprague-Dawley rats (aged 6 to 7 weeks; 120 to 150 g) were purchased from Shanghai Slac Laboratory Animal Co. Ltd. Ether inhalation was used to anesthetize the rats. N-methyl-nitrosurea solution (0.2 ml at 10 mg/ml; Sigma, St. Louis, MO, USA) was used to infuse the rat bladders via a 22-gauge angiocatheter every other week for five times. The animals remained anesthetized for approximately 45 min after catheterization to avoid spontaneous micturition (39).

Tumors were successfully induced at 16 weeks. Thereafter, 48 rats were assigned to four groups with 12 rats in each group. From the 16th week, and after anesthetizing the rats, their bladders were instilled with 500 μl of HNTs/siRIPK4, an equal dose of free HNTs, siRIPK4 only, or PBS solution. After instillation, the animals were kept under anesthesia for about 45 min to avoid spontaneous micturition. The treatments were repeated once a week for 5 weeks. Two days after the end of therapy, the animals were euthanized. Their bladders were removed, weighed, fixed in 4% PFA for 24 hours, embedded in paraffin, and analyzed histopathologically. Transverse sections were cut through the midportion of the bladder and subjected to H&E staining.

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