Zen 2 microscope software

Single cells were mixed in 0.3% agar (in DMEM-F12 supplemented with 10% FBS and 1% antibiotic/antimycotic) and plated at 1 × 10⁴ onto 6-well plates containing a solidified bottom agar layer (0.6% agar in the same growth medium). Cells were maintained at 37 °C and 5% CO₂ for 14 days. Colonies were photographed in 10 pattern fields, counted and measured using ZEISS ZEN 2 Microscope Software (ZEISS).

Fluorescence microscopy was used to investigate free sterol content and its cellular distribution by Filipin III staining (final concentration of 0.5 μg ml⁻¹) and neutral lipid content by Nile Red staining (final concentration of 2.5 μg ml⁻¹). For that, yeast cells from the parental and pdr18Δ strains harvested from cultivation at 30°C and 40°C were re‐suspended in PBS buffer to a final OD_600nm of 0.5. Cells were then incubated with each probe for 20 min, in the dark, in a tube rotator (VWR) and washed twice with PBS buffer. For cell‐to‐cell analysis, fluorescence was examined with an Axioplan microscope equipped with adequate epifluorescence interface filters, obtained from Zeiss. Fluorescence emission was collected with a coupled device camera (Axiocam 503 colour; Zeiss), and the images were analysed with ZEN 2 Microscope Software (Zeiss). The exposure time was kept constant among experiments for each probe, and intensity measurements were background‐corrected. For total quantification of the population's fluorescence, cell suspensions stained with either Filipin III or Nile Red were standardized to OD_600nm of 0.5 and the fluorescence was measured in a Filtermax F5 Microplate Reader (Ex/Em of 360/465 nm for Filipin III and 530/625 nm for Nile Red). Results were obtained from three independent biological replicates.