Cyclosporin a csa

The wild-type (WT) strain NRRL 8044 (ATCC 34921) of Tolypocladium inflatum (1 (link)) was used to generate the gene deletion mutants. Both the WT and mutants were maintained either on potato dextrose agar (PDA) (BD Difco) or in a Sabouraud dextrose broth (SDB) (BD Difco). For induction of CsA production, the WT or mutants was induced in the cyclosporine (CSN) induction medium containing fructose (fructose CSN induction medium) [fructose, 30 g/liter; (NH₄)₂HPO₄, 6 g/liter; yeast extract, 5 g/liter; CaCl₂·2H₂O, 1.32 g/liter; MgSO₄·7H₂O, 2.05 g/liter; FeSO₄·7H₂O, 27.4 mg/liter; ZnSO₄·7H₂O, 17.8 mg/liter; CoCl₂·6H₂O, 27.5 mg/liter; CuSO₄·5H₂O, 3.1 mg/liter] adjusted from a previous study (17 (link)). The strains of Aspergillus flavus (NRRL 3357), Metarhizium robertsii (ARSEF 23) (33 (link)), Cordyceps cicadae (CCAD02) (34 (link)), Cordyceps militaris (Cm01) (35 (link)), and Beauveria brongniartii (RCEF 3172) (36 (link)) were used for antifungal assays between WT and null mutants of T. inflatum. The standards of cyclosporin A (CsA) and d-Ala were ordered from Sigma-Aldrich (USA), and CsB and CsC were purchased from Santa Cruz Biotechnology (USA).

The imaging procedure followed a previously described method [18 (link)]. Cells were seeded on poly-d-lysine-coated glass coverslips and imaging was performed in Hank’s balanced salt solution (HBSS, Gibco, New York, NY, USA) at room temperature. To track changes in mitochondrial membrane potential, the fluorescent probe tetramethylrhodamine, methyl ester (TMRM, Invitrogen, Waltham, MA, USA) was used. Cells were incubated with 20 nM TMRM for 15 min in the dark at room temperature. Recordings were performed in the presence of 20 nM TMRM.
To induce mitochondrial Ca²⁺ overload and subsequent mPTP, ferutinin at a concentration of 20 μM was used. RI images (holographic reconstructions) and TMRM signals were acquired every 15 s using a 3D Cell Explorer-fluo (Nanolive, Tolochenaz, Switzerland) equipped with a 60× objective. At the end of each experiment, 10 μM of the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, Sigma Aldrich, St. Louis, MO, USA) was added to induce complete depolarization of mitochondria, allowing for the determination of the minimal TMRM signal for subsequent normalization and analysis.
Cyclosporin A (CSA, Sigma Aldrich, St. Louis, MO, USA) and bongkrekic acid (BA, Sigma Aldrich, St. Louis, MO, USA), inhibitors of mPTP and ANT, respectively, were used to explore the regulation of the mitochondrial permeability transition.

The cells were incubated with 10 μM rhodamine 123 (Sigma) in transport buffer (distilled water with 0.12 M NaCl, 25 mM NaHCO₃, 3 mM KCl, 2 mM MgSO₄, 2 mM CaCl₂, 0.4 mM K₂HPO₄, 1 mM HEPES, and 0.1% BSA) for 2 h at 37 °C. For inhibition experiments, cells were pre-incubated with 10 μM cyclosporin A (CsA; Sigma) for 60 min at 37 °C. Then, cells were washed three times with PBS and lysed with RIPA buffer. Fluorescence was measured using a fluorescence multi-well plate reader (Genios). Fluorescence was normalized on a per cell basis by counting dissociated cells.

EBV B95-8 virus was produced from the B95-8 Z-HT cell line, as previously described [36 (link)]. Buffy coats were obtained from normal donors through the Gulf Coast Regional Blood Center and peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll Histopaque-1077 gradient (Sigma, St. Louis, MO, USA #H8889). Primary human B cells were cultured in Roswell Park Memorial Institute medium (RPMI)1640 supplemented with 15% fetal bovine serum (FBS), 2 mM l-glutamine, penicillin, and streptomycin (1X, Sigma, #G6784) (R15) as well as 0.5 µg/mL Cyclosporin A (CsA) (Sigma, #30024). Bulk infections were performed by incubating cells with B95-8 Z-HT supernatant at 500 μL per 10⁶ B cells calculated from within the PBMC population for 1 h at 37 °C in a CO₂ incubator. Incubation was followed by washing in PBS and resuspending in R15 media supplemented with CsA. Bulk infections were conducted on 5 × 10⁸ PBMCs. LCLs were generated from normal donors by continuous outgrowth of EBV-infected primary B cells for greater than 35 days. LCLs were cultured in RPMI media supplemented with 10% FBS (R10). Bloom syndrome (BLM) deficient LCLs (GM16377, GM16375, and GM09960) were obtained from Coriell Institute (Coriell Institute, Camden, NJ, USA).