N methyl n nitroso urea nmu

Mice were injected intratracheally (i.t.) with N-methyl-N-nitroso-urea (NMU; Sigma-Aldrich, Rome, Italy) for three consecutive weeks at the dose of 50 μg/mouse (day 0) followed by other two administrations of 10 μg/mouse (day 7 and 14) (Supplementary Figure 1A) [8 (link)]. Mice were sacrificed at day 28, four weeks post the first administration of NMU. Broncho-alveolar lavage fluid (BAL) was collected using 0.5 ml of PBS containing 0.5 mM EDTA and cell counts performed.

According to the previous studies to induce the prostate cancer in the cancerous groups, the animals received a daily dose (50 mg/kg body weight) of cyproterone acetate (Sigma-Aldrich, St Louis, MO, USA) as intraperitoneal injections for 18 consecutive days. cyproterone acetate (CA) was used to prevent release of androgens from testis and to induce atrophy in the prostate epithelium. On day after the last injection of CA, the animals received a daily subcutaneous injection (100 mg/kg) of testosterone propionate (Sigma-Aldrich, St Louis, MO, USA) for three days to stimulate proliferation of prostate epithelium. On day after the last injection, the N-Methyl-N-nitrosourea (NMU, Sigma-Aldrich, St Louis, MO, USA) was injected to the animals as intraperitoneal at a dose of 50 mg/kg. The NMU powder was first wetted with 3 % acetic acid and then diluted with saline to prepare a final concentration 10 mg/mL with pH 5.5 for injection (Bosland and Prinsen, 1990[7 (link)]; Arroyo-Acevedo et al., 2017[3 (link)]).