2 tert butyl 1 4 benzoquinone

C. albicans strains were grown overnight in YPD at 30°C diluted to 2.5 × 10⁵ cells and then spread onto the surface of a synthetic complete medium agar plate. Ten microliters of each compound was applied to paper filter disks (Becton, Dickinson and Company, Sparks, MD), the disks were applied to the plate surface, incubated at 30°C for 48 h, and then the diameter of the zone of growth inhibition (halo) was measured. Compounds tested included p-benzoquinone (Sigma-Aldrich, St. Louis, MO), menadione (Sigma-Aldrich, St. Louis, MO), 2-tert-butyl-1,4-benzoquinone (Cayman Chemical, Ann Arbor, MI), hydrogen peroxide (Sigma-Aldrich, St. Louis, MO), tert-butyl-hydroperoxide (tBOOH; Acros Organics) and diamide (Sigma-Aldrich, St. Louis, MO). For the CFU assays, wild-type and the 3×GFP-tagged strains were grown overnight, harvested by centrifugation, and resuspended in PBS. A total of 1 × 10⁷ cells/mL were inoculated in liquid YPD and treated with one of the following: 100 µM of p-benzoquinone, 100 µM 2-tert-butyl-1,4-benzoquinone, 100 µM menadione, or 500 µM of hydrogen peroxide. Tubes were incubated for 1 h at 30°C on a tube roller, after which cultures were centrifugated and cells washed twice with PBS. Serial dilutions were plated on YPD plates, incubated for 48 h at 30°C, and then colony-forming units were counted.

3×GFPγ tagged C. albicans strains were grown overnight in YPD at 30°C and then diluted 1:250 in 5 mL YPD and grown until a density of about 1 × 10⁷ cells/mL. Cells (1 mL) were treated with 100 µM of one of the quinones p-benzoquinone (Sigma-Aldrich, St. Louis, MO), 2-tert-butyl-1,4-benzoquinone (Cayman Chemical, Ann Arbor, MI), menadione (MND; Sigma-Aldrich, St. Louis, MO) or 500 µM of H₂O₂ and incubated for 1 h at 30°C on a tube roller. Samples were centrifuged, washed in sterile phosphate-buffered saline (PBS), and analyzed by fluorescence microscopy. To assess the effect on Zta1 production at different times and concentrations of incubation with BZQ, cells were treated with 10, 30, or 100 µM of BZQ, or treated with 100 µM of BZQ for 15, 30, and 60 minutes of incubation and then prepared for imaging as described above. Zeiss ZEN software was used to control the microscope and for deconvoluting images and calculating the mean fluorescence intensity (MFI) of cells. Statistical analysis of MFI compared to non-treated GFP-tagged cells was conducted with Prism 6 software (GraphPad Software, Inc., La Jolla, CA).