![\"Creative](\"https://i.creativecommons.org/l/by/4.0/88x31.png\")
*Detailed protocol:
\r\n**1. Epifluorescence microscopy: C. albicans cells expressing Ato-GFP fusion proteins (wild-type and ato-mutants) were grown in a SM medium supplemented with 0.2% glucose until the exponential phase at 30 °C, 200 rpm. Cells were then washed and transferred to SM media containing acetic acid (0.1% v/v, pH 6.0), lactic acid (0.1% v/v, pH 5.0), or without carbon source (YNB) at 30 °C, 200 rpm. Samples were collected at 0, 5, and 24 h after derepression for analyses by epifluorescence microscopy (n=3). In addition cells expressing Ato-GFP fusion proteins were grown directly in SC medium containing different carboxylic acids: formic acid (0.1% v/v, pH 6.0), butyric acid (0.1% v/v, pH 6.0), succinic acid (0.1% w/v, pH 6.0), pyruvic acid (0.1% w/v, pH 6.0), malic acid (0.1% w/v, pH 6.0), citric acid (0.1% w/v, pH 6.0) and 0.2% (w/v) glucose at 30 °C 200 rpm. Cells were visualized by fluorescence microscope after 16, 18, 20, 24, and 40 h of growth in the specified medium (n=2). The time point t≈16 h corresponded to the beginning of the exponential phase (0.4 < OD640 nm < 0.6). For microscopy analysis, 1 mL of each culture was harvested, cells were concentrated by centrifugation and manually immobilized on slides. Cells were visualized using a Leica DM5000B microscope with appropriate filters. Images were captured with a Leica DFC 350FX R2 digital camera using LAS AF V1.4.1 software (Leica).
\r\n**2. Cell staining with DAPI: C. albicans Ato2-GFP and Ato2-GFP ato1Δ/Δ strains were grown in a SM medium (0.2% glucose) to mid-log phase (OD₆₄₀ = 0.5). Cells were harvested, washed twice with deionized water, and transferred to SM media containing acetic acid (0.1% v/v, pH 6.0). After 5 h of derepression, 1 ml of cells was collected and resuspended in 1 ml 3.7% paraformaldehyde (PFA), then incubated 1 h at room temperature with shaking at 200 rpm. After fixation, cells were washed three times with phosphate-buffered saline (PBS) and resuspended in 1 ml PBS. DAPI nuclear stain was added to a final concentration of 2 μg/mL, and cells were incubated at room temperature for 10 min with shaking at 200 rpm. Following two additional washes with PBS, cells were visualized using a Leica DM5000B microscope with appropriate filters. Images were captured with a Leica DFC 350FX R2 digital camera using LAS AF V1.4.1 software (Leica) (n=2).
\r\n**3. Western Blot : Yeast cells were grown as explained above in section 1. (Epifluorescence microscopy). At the time-point 5 h, cells were harvested at a final OD640 of 1.5 per mL, and total protein extracts were prepared using the NaOH/TCA method. Briefly, cells were harvested by centrifugation (max. rpm, 1 minute (min), 4 °C) and the pellet was resuspended in 500 µL of cold water. Cell lysates were prepared by adding 50 µL of NaOH (1.85 M) following 10 min of incubation on ice. Then, 50 µL of trichloroacetic acid (TCA, 50%) was added and the samples were incubated for an additional 10 min on ice. Protein precipitates were collected by centrifugation (max. rpm, 15 min, 4 °C). The pellets were dissolved in 50 µL of loading buffer (33.3 mM Tris-hydrochloride pH 6.8; 1.33 mM ethylenediaminetetraacetic acid (EDTA); 1.33% sodium dodecyl sulphate (SDS); 6.66% glycerol; 0.02% bromophenol blue and 2% beta-mercaptoethanol) and heated at 37 °C for 10 min. Each sample (10 µL) was loaded onto a 10% acrylamide gel. Following electrophoresis, the separated proteins were transferred onto nitrocellulose membranes (GE Healthcare Life Sciences. For transference and loading controls, the membranes were stained with a Ponceau S solution (0.1% Ponceau (w/v); 0.5% glacial acetic acid (v/v)). Following washing and blocking steps, the membranes were probed with the primary antibody anti-GFP (monoclonal, mouse IgG1κ, clones 7.1 and 13.1, Roche, 11814460001) used at 1:3000 dilution. The antimouse-IgG (whole molecule)-peroxidase produced in rabbit (A9044, Sigma) was used as a secondary antibody at 1:100000 dilution. The signal was detected by enhanced chemiluminescence using the WesternBright ECL HRP substrate (advansta). All images were captured using a Syngene G: Box Chemi XX9 image documentation system and GeneSys software (n=3).
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P.; Van Dijck, Patrick; Pittis, Alexandros A.; Diallinas, George; Soares-Silva, Isabel; Paiva, Sandra, 2026, \"Expansion, functional diversification and gene fusion events in the Ato protein family\", https://doi.org/10.34622/datarepositorium/EQT3Q4, Repositório de Dados da Universidade do Minho, V1"}}