Nile Red is excluded from the tif51A-1 mutant because of high Pdr5 pumping activity. (A) Wild-type strain and tif51A-1 were cultured in SGal medium at 25°C until reaching post-diauxic phase and then transferred to 25°C or 37°C for 4 h. Then, cells were incubated with Nile Red substrate for 15 min prior to microscopy. A representative image is shown from three independent experiments. Scale bar, 4 μm. (B) Quantification of Nile Red fluorescent signal from at least 150 cells. (C–F) Wild-type strain and tif51A-1 expressing Tom70-GFP (C), Yta12-GFP (D), Cyc1-GFP (E), or Ilv2-GFP (F), and Su9-mCherry were cultured in SGal medium at 25°C until reaching post-diauxic phase and subjected to fluorescence microscopy. A representative image is shown from three independent experiments. Scale bar, 4 μm. Data information: In B, results are presented as mean ± SD from three independent experiments. The statistical significance was measured by using a two-tailed paired Student’s t test. ***P < 0.001. n.s means no significant differences.