For cell morphology, cells were grown in YPD to early log phase from YPD overnight cultures. Samples were taken, washed and resuspended in PBS www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html buffer, and sonicated for 5 seconds at 30% amplitude in a Fisher Scientific 150T Series Sonic Dismembrator (Fisher Scientific, USA). Light microscopy was used to quantify numbers of single unbudded cells, budding cells, and cells with abnormal or pseudohyphal-like morphology. To assess nuclear integrity, cells were grown to early log phase and stained with DAPI according

to a previously published protocol [35]. Overnight cultures were diluted to an OD600 of 0.05 in 5 mL of YPD and allowed to grow for 4 hours at 30°C. Samples were spun down, washed EVP4593 solubility dmso in 1 mL of 1X PBS, and fixed overnight at 4°C in 1 mL of 70% ethanol. Fixed cells were washed and treated for 2 hours in 55 mM HCl with 5 mg/mL pepsin at 37°C, then washed and resuspended in

1 mL of 1X PBS containing 2.5 μg/mL DAPI (Sigma-Aldrich, Akt inhibitor St. Louis, MO, USA). Cells were sonicated and visualized using a Zeiss Axio ImagerZ.1 microscope (Carl Zeiss Microimaging, Inc, Thornwood, NY, USA). DNA damage and antifungal drug sensitivities To test the sensitivity of strains in this study to various agents, the agar spot dilution method was used. Overnight YPD cultures were diluted to an OD600 of 1.0 and serial ten-fold dilutions were made to 10-6. 2 μL volumes of each dilution were spotted onto YPD plates, and YPD plates containing FLC, MMS or menadione (Sigma-Aldrich, St. Louis, MO, USA) at the indicated concentrations.

Plates were incubated for 48 hours at 30°C and images were taken. E-test analysis was performed for common antifungals, using overnight cultures diluted to an OD600 of 0.05 to spread a lawn on CAS plates (9.0 g casitone, 5.0 g yeast extract, 0.54 g KH2PO4, 3.34 g K2HPO4, 20. 0 g dextrose and 20.0 g agar per liter). E-test strips were placed on plates, which were incubated for 48 hours at 30°C. Two independent nulls of the RAD54 gene were tested. The MIC was read as the point at with the zone of inhibition intersected the E-test strip. Acknowledgements and Funding This work was supported by Public Health Service grants GM53738 (HLK), T32AI007180 (SJH) find more from NIAID, and DE17078 (TCW). We thank David Kirkpatrick of the University of Minnesota for kindly providing the MRE11, RAD50 and RAD52 mutant strains. References 1. Slavin MA, Sorrell TC, Marriott D, Thursky KA, Nguyen Q, Ellis DH, Morrissey CO, Chen SC: Candidaemia in adult cancer patients: risks for fluconazole-resistant isolates and death. J Antimicrob Chemother 2010, 65:1042–1051.PubMedCrossRef 2. Chen CG, Yang YL, Shih HI, Su CL, Lo HJ: CaNdt80 is involved in drug resistance in Candida albicans by regulating CDR1. Antimicrob Agents Chemother 2004, 48:4505–4512.PubMedCrossRef 3.