FIG 5 .

Homeostasis of ergosterol biosynthesis is required for the gamma radiation resistance of C. neoformans. (A) Expression analysis of ERG genes at the recovered time after exposure to radiation. The fold increase of ERG gene expression was quantitatively monitored using qRT analysis with the gene-specific primers listed in Table S2. Duplicate technical experiments with two biological samples were performed. Representative images from independent experiments for each ERG gene are shown. Error bars indicate standard deviations. Asterisks represent statistical significance of differences in expression levels of each ERG gene (**, P < 0.01; ***, P < 0.001). (B) Reduced ergosterol increased in the gamma radiation resistance in C. neoformans. C. neoformans WT (H99) strains were grown in a liquid medium at 30°C for 16 h, and then the grown cells were subcultured for 5 h to an OD₆₀₀ of 1.0. Cells were treated with or without fluconazole (10 µg/ml) for 90 min. Next, cells treated with or without fluconazole were exposed to the indicated dose of gamma radiation for 1 h. After radiation, cells were spread onto the solid YPD medium and further incubated at 30°C for 2 days. The relative survival rate was measured as the viable cell count number at the indicated dose of radiation divided by the viable cell count number before radiation. The relative survival rates were statistically compared between cells treated with or without fluconazole using the Bonferroni selected comparison test performed with Prism software, version 5.0 (GraphPad Software, Inc.). Asterisks represent statistical significance of differences in the relative survival rate (*, P < 0.05). (C) Ergosterol homeostasis is required for gamma radiation resistance. C. neoformans WT (H99), sre1Δ (YSB2493 and YSB2494), and hob1Δ (YSB2308 and YSB2309) mutants grown overnight were 10-fold serially diluted (1 to 10⁴) and then spotted onto the YPD medium. Cells were exposed to the indicate dose of gamma radiation for 1 h and then further incubated at 30°C for 3 days.