Paraformaldehyde (pfa)

12 µg/ml of H5K1 was added to different concentrations of laminarin in PBS 3% BSA in order to reach the following laminarin:H5K1 ratios: 40:1, 16:1, 4:1, 1:1 and 0:1. The solutions were left react at 37 °C for one hour then were put in contact with 3.0 × 10⁶C. auris cells coming from an overnight inoculum. After 1 h the cells were centrifuged, washed, and marked for 1 h with anti-human IgG FITC antibody (Abcam, ab97224) 1:150 in PBS + 3% BSA. The cells were washed before and after fixing with paraformaldehyde (Carlo Erba) 4% for 1 h at 4 °C and finally they were resuspended in 100 µl of PBS. The samples were analysed through immunofluorescence microscope.

Cells were fixed with 4% paraformaldehyde (Carlo Erba, Milan, Italy), permeabilized by 0.5% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA) and incubated for 1 h at 4 °C with the following antibodies, at a final concentration of 0.1 mg/mL: anti-AMOT1 (Santa Cruz Biotechnology, Inc. Dallas, TX, USA; sc-166924), anti-YAP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or anti-p-YAP (Cell Signaling Technology). For F-actin detection, cells were stained with Biotin-XX Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA; #B7474). After washings, cells were incubated for 30 min at 37 °C with a secondary antibody conjugated with Cy5 (Abcam) or Streptavidin-Cy5 (Thermo Fisher Scientific, Waltham, MA, USA). Cell samples were washed twice in PBS and immediately acquired by a cytometer.
For flow cytometry studies, samples were acquired with a FACScalibur cytometer (BD Biosciences Inc., San Diego, CA, USA) equipped with a 488 nm argon laser and with a 635 nm red diode laser. At least 20,000 events were acquired, recorded and analyzed using CellQuest software (BD Biosciences, San Diego, CA, USA). The expression level of the analyzed proteins by flow cytometry was reported as median fluorescence.

To analyze planktonic cells, the strain was inoculated as described above and aliquots were withdrawn after 2, 4, 6, and 8 h using a sterile 10 µL loop and a drop was placed on a microscope glass slide (SuperFrost, Thermo Scientific, Rodano, Italy). The drop was then fixed by covering with paraformaldehyde 4% (Carlo Erba) and dried on a hot plate at 50 °C. Cells were then stained with crystal violet (Carlo Erba) and analyzed by an upright optical microscope (Olympus CX43) with a 40× objective. For each time point, three drops were withdrawn from each tube. The experiment was performed in triplicate for each strain. Five random images were acquired from each sample.

From an overnight inoculum, microorganism cells or conidia were washed with RPMI + MOPS (0.165 M, pH 7) and 3.0 × 10⁶ cells were pelleted, resuspended in Phosphate buffered saline (PBS) containing 3% (w/v) BSA (Bovine Serum Albumin Sigma Aldrich) and put in contact with 12 µg/ml of the H5K1 for 1 h at room temperature. The cells and the conidia were washed with PBS and marked with anti-human IgG FITC antibody (Abcam, ab97224) 1:150 in PBS + 3% BSA for 1 h. The cells and conidia were washed and fixed with paraformaldehyde (Carlo Erba) 4% in PBS 1 h at 4 °C. After fixing and washing with PBS, the pellet was resuspended in 400 µl of PBS and splitted in two tubes. The samples of Candida auris were analysed respectively using flow cytometry and immunofluorescence while the others just in immunofluorescence^{47 (link)}. A Flow cytometer (FACScanto II, BDBioscences, Erembodegem, Belgium), equipped with three lasers (488 nm, 633 nm, 405 nm) was employed to collect and quantitate FITC fluorescence from different samples. Both autofluorescence and fluorescence derived from aspecific binding of FITC-conjugated secondary Ab were quantitated by flow cytometry.

Control (untreated or mCNF1‐treated) and CNF1‐treated fibroblasts, seeded on glass coverslips, were fixed with 3.7% paraformaldehyde (Carlo Erba), permeabilized with Triton 0.5% X‐100 (Sigma‐Aldrich, St. Louis) and then incubated at 37°C for 30 min with Tetramethylrhodamine‐isothiocyanate (TRITC)‐phalloidin (Sigma‐Aldrich, cat. n. P1951), diluted 1:100 in PBS for actin cytoskeleton staining. Nuclei were stained for 5 min at RT with 0.2 μg/ml Hoechst 33258 (Sigma‐Aldrich, cat. n. 94,403). To stain mitochondria, cells were incubated with 1 μM Mitotracker Red CMXRos (Invitrogen, Waltham, cat. n. M7512), 30 min before fixation with paraformaldehyde. To stain p62, cells were incubated with Anti‐p62/SQSTM1primary antibody (Sigma‐Aldrich, cat. n. P0067) for 30 min and, following extensive washing, incubated with the appropriate secondary antibody (Alexa Fluor Thermofisher Scientific Invitrogen, cat n. A‐11034). Finally, glass slides were observed with an Olympus BX51/BX52 fluorescence optical microscope (Olympus Corporation of the Americas, Center Valley) equipped with a charge‐coupled device camera (Carl Zeiss). Images were acquired using the IAS 2000 (Delta Systems Inc., Streetsboro) programme.

Control and treated cells were fixed with 4% paraformaldehyde (Carlo Erba, Milan, Italia) and then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). After washings, cells were incubated with the following monoclonal antibodies (mAb) or polyclonal antibodies (pAb), alone or in combination, for 1 h at 37 °C: mAb anti-LAMP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and pAb anti-LC3 (MBL International, Woburn, MA, USA). After washings, cells were incubated with anti-mouse AlexaFluor 594-conjugated and anti-rabbit AlexaFluor 488-conjugated antibodies (all Thermo Fischer Scientific, Waltham, MA, USA) for additional 30 min at 37 °C. All samples were counterstained with Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) and mounted with Dako Fluorescent Mounting Medium (Dako, Santa Clara, CA, USA). The images were acquired by IVM with an Olympus fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA, USA), equipped with a Zeiss charge-coupled device camera (Carl Zeiss, Oberkochen, Germany).
For mitochondrial staining, cells were incubated with 1 µM Mitotracker Red CMXRos (Invitrogen, Carlsbad, CA, USA) for 1 h before fixation and then processed for fluorescence microscopy.

Control and treated cells were fixed with 4% paraformaldehyde (Carlo Erba, Milan, Italy) and then permeabilized by 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). After washings, cells were incubated with PAb to TOM20 (Santa Cruz Biotechnology Inc., Dallas, TX, USA). After washings, cells were incubated with antirabbit AlexaFluor 594-conjugated (Termo Scientific, Rockford, IL, USA) for additional 1 h at 37 °C. All samples were counterstained with Hoechst 33342 (Termo Scientific, Rockford, IL, USA) and mounted with glycerol-PBS (2:1). The images were acquired by intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA, USA), equipped with a Zeiss charge-coupled device (CCD) camera (Carl Zeiss, Oberkochen, Germany).