Cortisone acetate

For the immunocompromised mouse model, we used outbred CrlOri:CD1 (ICR) (Orient Bio Inc, Korea) female mice (30 g in body weight, 6 to 8 weeks old), which were housed five per cage and had access to food and water ad libitum. Mice were immunosuppressed with subcutaneous injections of cortisone acetate (Sigma-Aldrich, USA) at 10 mg/mouse for 4 days prior to infection and cortisone acetate injected subcutaneously, 10 mg/mouse, 2 days prior to infection. At days 0, 3, and 6 post-infection, administrations were repeated with cortisone acetate (10 mg/mouse). For conidia inoculation, mice were anesthetized with isoflurane, and then intranasally infected with 1 X 10⁷ conidia of A. fumigatus strains (10 mice per fungal strain) in 30 µL of 0.01% Tween 80 in PBS. Mice were monitored every 12 h for survival for 8 days after the challenge. Mock mice included in all experiments were inoculated with sterile 0.01% Tween 80 in PBS. Mice were checked every 12 h for survival and Kaplan–Meier survival curves were analyzed using the log-rank (Mantel–Cox) test for significance (p < 0.01).

Seven-week-old female BALB/c mice weighing 18–20 g (The Jackson Laboratory) were used for the invasive pulmonary aspergillosis model^{18 (link)}. Immunosuppression was achieved by injection of cyclophosphamide (Baxter Healthcare) intraperitoneally 4 days at 150 mg/kg and 1 day at 100 mg/kg before inoculation and injection of cortisone acetate at 250 mg/kg (Sigma) subcutaneously 1 day before inoculation. On inoculation day, 40 μL of A. fumigatus conidial suspension containing 5 × 10⁴ freshly collected conidia in sterile PBS + 0.02% Tween 20 were intranasally inoculated under anesthesia. Additional doses of cyclophosphamide (100 mg/kg) were given on days 2 and 6 after inoculation to maintain neutropenia. Mortality was assessed daily until day 14 after inoculation. Animals displaying signs of morbidity were euthanized, and their death was recorded as occurring 12 hours later^{19 (link)}. 200 μL of 10 mg/mL doxycycline were treated twice a day via gastric lavage. Statistical analyses for comparison of survival rate between WT and knockout, or between P_Tet-Off-target gene with doxycycline and without doxycycline were conducted using Prism 6 and p values from the Log-rank (Mantel-Cox) test were presented.