Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

Reverse transcriptase “Superscript III” (Invitrogen, 18080-051) was used to perform cDNA synthesis. Briefly, 500 ng of DNase I-digested RNA was incubated with 1 μl of 10 mM dNTP and 50 μM oligo(dT) at 65°C for 5 min in a 10 μl reaction mixture followed by cooling on ice for 5 min. Post incubation, 10 μl of cDNA synthesis mixture was added which contained 2 μl of 10XRT buffer, 4 μl of 25 mM MgCl2, 2 μl of 0.1 M DTT, 1 μl of RNase out (40 units) and 1μl of Superscript III (200 units). Tubes were incubated at 50°C for 1 h and thereaction was terminated at 85°C for 5 min. The quality of synthesized cDNA was checked by using it as a template in a PCR reaction to amplify the housekeeping gene CgACT1. Amplification of CgACT1was indicative of proper cDNA synthesis

Complementary DNA (cDNA) synthesis

ml YPD broth at an initial OD600of 0.1. Cultures were allowed to grow for 4-5 hin a shaker incubator setat 30°C, 200 rpm until the OD600of the cultures reached 0.4-0.6. Next,cells were harvested ina15 ml centrifuge tube by centrifugation, washed twice with 10 ml of sterile water, resuspended in 1 ml of sterile water and were transferred to a 1.5 ml microfuge tube. Cells were harvested by centrifugation at 4,000 rpm for 5 min,resuspended in 50 μl of100 mM lithium acetate solution and transformation mixture was added. Transformation mixture consisted of 240 μl polyethylene glycol (50%), 36 μl of lithium acetate (1 M), 5 μl of heat denatured single stranded carrier DNA (10 mg/ml), 500 ng to 1 μg of transforming DNA and final volume was made to 360 μl with sterile water. The tubes were incubated at 30°C for 45 min. To this, 43 μl of sterile DMSO was added and heat shock was given at 42°C for 15 min. Next, tubeswere transferred to ice for 10-15 sec, centrifuged at 4,000 rpm and transformation mixture reagents wereremoved completely by pipetting. Cells were resuspended in 200 μl of sterile water and spread-plated on appropriate selection medium. Plates were incubated at 30°C for 24-48 h

Yeast transformation was performed as described previously (Gietz et al., 1992) with fewmodifications. Briefly, overnight grown C. glabratacultures were freshly inoculated in 10

Yeast transformation

To perform survival analysis of C. glabratacells in macrophages, PMA-treated THP-1 cells were seeded to 24-well tissue culture platesto afinal celldensity of 1 millionper well. C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 mloftheseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x106cells/ml. 50 μl of this cell suspension was used to infect PMA-activated macrophages to a MOI (multiplicity of infection) of 0.1. Two hours post infection, THP-1 cells were washed thrice with 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed complete RPMI-1640 medium was added.At different time points, infected THP-1 macrophages were osmolysed with1 ml sterile water. Post lysis,lysates were collected by scraping the wells using 1 ml microtip. Lysates were diluted in 1X sterile PBSand appropriate dilutions were plated onYPD-agar plates. Plates were incubated at 30°C for 24-48 h and colony forming units (CFUs) were counted. Final CFUs/ml were determined by multiplying CFUs withappropriate dilution factor and percentage phagocytosis was calculated by dividing CFUs obtained at 2 h post infection by total numberofyeast cells used for infection. Fold replication was calculated by dividing the CFUs obtained at 24 h post infection by CFUs obtained at 2 h post infection

THP-1 macrophageinfection assay to monitor the intracellular survival and replication of C. glabrata

Cell viability can be monitored by changes in the morphology of the cell or by membrane permeability to certain dyes such as PI. When exposed to PI, viable cells do not take up PI due to the presence of an intact membrane whereas dead cells take-up PI due to loss of membrane integrity. Briefly, cells were treated with HU for 12 h, and allowed to recover for different lengths of time as described in Section 2.2.2. At each time point, cells were harvested by trypsinisation and washed with PBS. Approximately 106cells were resuspended in 1 ml PBS and stained with 2μg/mL (final concentration) PI for 2 min at room temperature. The extent of PI staining was used to determine the viable and dead population by flow cytometry analysis (Accuri C6, Becton Dickenson). Nonviable cells were approximately 100 fold brighter than the unstained (viable cells) cells

PI live dead analysis