T7660

To instigate oral candidiasis in the mice, a specific treatment and infection protocol were implemented. The animals underwent immunosuppression through two subcutaneous injections of prednisolone (Sigma Aldrich-P6004, Taufkirchen, Germany) at a dosage of 100 mg/kg. These injections were administered one day prior to and three days following the infection with Candida albicans ATCC 10231. Additionally, tetracycline hydrochloride (Sigma Aldrich-T7660, Taufkirchen, Germany) was introduced into the mice’s drinking water at a concentration of 0.9 mg/ml, starting one day before the infection. Anesthesia was induced through intramuscular injections of 2 mg/ml chlorpromazine chloride (Sigma), with 50 μl administered on each femur.
To initiate oral infections, small cotton pads (baby cotton buds from Johnson & Johnson) were immersed in a cell suspension of C. albicans ATCC 10231 containing 2.0 x 10⁸ viable cells per milliliter. These saturated cotton pads were then used to gently swab the entire oral cavity of the anesthetized mice. The daily assessment of infection severity relied on monitoring the presence and intensity of whitish, curd-like patches observed on the surface of the tongue [16 (link)].

Hydroxyurea (3 mM for 2 h for QIBC and immunoblotting, 0.5 mM, 1 mM, or 2 mM for 24 h for clonogenic assays, H8627; Sigma-Aldrich), UCN-01 (300 nM for 1 h, U6508; Sigma-Aldrich), APH (50 μg/ml for 2 h, A4487; Sigma-Aldrich), thymidine (2.2 mM for 12 or 17 h, T1895; Sigma-Aldrich), nocodazole (100 ng/ml for 12 h, M1404; Sigma-Aldrich), PD0332991 (5 μM for 12 h, PZ0383; Sigma-Aldrich), TAK-931 (300 nM for 12 h, HY-10088; Biotech), bleomycin (25 μg/μl for 3 h on and 3 h off, S1214; Stratech), CPT (500 nM for 6 h, C9911; Sigma-Aldrich), Z-VAD-FMK (50 μM, ab120487; Abcam). Protein expression was induced in with 0.5–1 μg/ml tetracycline for 24 h (T7660; Sigma-Aldrich).

To measure intracellular Ca²⁺ concentrations ([Ca²⁺]_i), P2X7R-HEK cells were seeded in glass bottom culture dishes (CELLview™, Greiner Bio-One, Kremsmünster, Austria) and P2X7R expression was induced by the addition of 1 μg/mL tetracycline (Sigma-Aldrich T7660) and culturing at 37 °C, 5% CO₂, for 24–48 h. Thereafter, the culture medium was replaced by the same bath solution as described for the whole-cell patch-clamp recordings. Measurements were performed as described previously [11 (link)]. In brief, P2X7R-HEK cells were loaded with 3.3 µM Fura-2/AM (Thermo Fisher Scientific) for 25 min at 37 °C. Fura-2/AM was excited at 340 and 380 nm wavelengths and the fluorescence emission was measured at 510 nm. Three independent batches of P2X7R-HEK cells were used in these experiments and a total number of 411 cells were tracked individually. The fluorescence intensity ratio of 340:380 nm was recorded. All experiments were performed at room temperature.