Nystatin nys

RK-13 cells were pretreated for 30 min at 37°C with each of the inhibitors followed by cold-synchronized infection. Where indicated, inhibitors were added to previously infected cells at 2 or 3 hpi. Stock solutions of 50 mM nystatin (Nys) (Sigma-Aldrich Corporation) and 50 mM 5-ethylisopropyl amiloride (EIPA) (Sigma-Aldrich Corporation) were dissolved in DMSO. Water was used as a solvent for 10 mM chlorpromazine (CPZ) (Sigma-Aldrich Corporation). All inhibitors were present throughout the experiments. This study was required given that we used the highest doses reported in the literature to ensure the efficacy of the inhibitors. We also assessed the cytotoxic activity of the organic solvent DMSO. Based on the results of these experiments, optimal non-toxic working concentrations were selected for the internalization assay. For the blocking experiments, RK-13 cells were pretreated in serum-free minimal essential medium containing DMSO as a control, 100 mM Nys, 50 mM EIPA, or 30 mM CPZ. These drug-treated cells were subsequently infected with RHDV at a MOI of 1, incubated in growth medium for 1 h at 37°C, and harvested for flow cytometry or qRT-PCR analysis.

A total of twenty-four, 6 day-postpartum (dpp) male Swiss albino mice were used in the study. All experiments and animal handling were conducted in accordance with the institutional guidelines for animal experimentation after obtaining prior approval from the Institutional Animal Ethics Committee (Kasturba Medical College & Kasturba Hospital Institutional Ethics Committee, approval #IAEC/KMC/93/2013). Animals were sacrificed by cervical dislocation and the testes were collected in alpha minimum essential medium (α-MEM + Glutamax; (32571-036; Gibco™, Grand Island, USA) containing 1% (v/v) penicillin-streptomycin (Pen-Strep; 15140-122; Gibco™, Grand Island, USA) and 5 µg/mL Nystatin (Nys; N3503; Sigma-Aldrich, St. Louis, USA). Testes were made fat-free using fine needles, under the stereomicroscope, and later randomly distributed/categorized for either holding-phase or direct culture.

KCl, NaCl, HEPES, EDTA, nonactin, pentane, ethanol, methanol, calcein, triton X-100, sephadex G-50, digitonin, tribulosin, dioscin, diosgenin, solasodine, escin, uvaol, lupeol, betulin, nystatin (NyS), and gramicidin A (GrA) were purchased from Sigma-Aldrich (St. Louis, USA). Syringomycin E (SyrE) was isolated and purified as described previously [37 (link)], and kindly offered by Dr. J.Y. Takemoto (Utah State University, USA). The chemical structures of tested agents are presented in Figure 6. The purity of saponins and related compounds was ≥95% (except for betulin (≥98%), lupeol (≥94%), diosgenin (≥93%), and digitonin (~50%)). All experiments were performed at room temperature (25 °C).
Lipids, 1,2-diphytanoil-sn-glycero-3-phosphocholine (DPhPC), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine (HOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DPPG), and cholesterol (CHOL) were obtained from Avanti Polar Lipids (Pelham, NY, USA).