FIG 8.

Vacuole morphology and pH are affected in yeast old cells. (A and D) Images depicting vacuolar morphology (white arrows) stained with FM4-64 in young (Y) and old (O) cells of C. neoformans (Cn) (A) and C. glabrata (Cg) (D). DIC, differential interference contrast. (B and E) The ratio of vacuolar/cell area was significantly increased in old (red) Cn (B) and Cg (E) compared to younger cells (blue). An unpaired t test with Welch’s correction was used to generate the P-values (*, P = 0.0245; ****, P < 0.0001). (C and F) The average number of vacuoles per yeast cell was significantly lower in old (red) Cn (C) and Cg (F) compared to the respective young cells (blue). An unpaired t test with Welch’s correction was used (**, P = 0.0098; ****, P < 0.0001). For all analyses, 25 cells of each group were measured using ImageJ/Fiji software. Measurement of quinacrine levels with a spectrophotometer showed a significant decrease in fluorescence in aged Cn compared to young cells (G). This was confirmed with microscopy. In contrast, significantly increased fluorescence was measured in old cells of Cg compared to the young cells (H). This was also confirmed with microscopy. The assay was done in biological triplicate and an unpaired t test with Welch’s correction was used to generate the P values (*, P = 0.0155; ***, P = 0.0007).