FIG 5.

CgSet2 is a histone H3 lysine 36 methyltransferase. (A) Serial dilution spot analysis illustrating the fluconazole susceptibility of the Cgrph1Δ and Cgset2Δ mutants. Visible artifacts appear within the panels upon overexposure. (B) Representative immunoblots showing H3K36me3 modification levels in the indicated C. glabrata strains. Whole-cell lysates of YPD-grown log-phase cultures were prepared, and 50 μg protein was resolved on a 15% SDS-PAGE gel and probed with anti-H3K36me3, anti-H3, and anti-GAPDH antibodies. GAPDH serves as a loading control. For quantification, the intensity of individual bands from 3 to 4 independent Western blots was measured using ImageJ densitometry software. The histone H3K36me3 signal was normalized to the corresponding total histone H3 signal for each strain. Data (means ± SEM; n = 3 to 4) represent the percent changes in histone H3K36me3 levels in mutants compared to the wt strain (considered 100%) and are presented as a bar graph on the right side of the blot image. ****, P ≤ 0.0001 (by paired two-tailed Student’s t test). (C) qPCR-based analysis of CgPDR1, CgCDR1, and CgCDR2 transcript levels in the wt, Cgset2Δ, and Cgrph1Δ strains. Data (means ± SEM; n = 3 to 4) were normalized against the CgTDH3 mRNA as a control and represent fold changes in the expression of the CgPDR1, CgCDR1, and CgCDR2 genes in the Cgset2Δ and Cgrph1Δ mutants, compared to the wt strain (considered 1.0). *, P ≤ 0.05; **, P ≤ 0.01 (by paired two-tailed Student’s t test). (D) Organ fungal burden in BALB/c mice infected intravenously with wt, Cgset2Δ, or Cgrph1Δ C. glabrata cells. After 7 days of infection, the fungal burden in the indicated mouse organs was determined by organ collection and homogenization, followed by homogenate plating onto YPD medium containing penicillin and streptomycin. Diamonds and bars represent CFU recovered from organs of the individual mice and the CFU geometric mean (n = 6 to 8) for each organ, respectively. **, P ≤ 0.01; ***, P < 0.001 (by a Mann-Whitney U test).