Zen 2011

Confocal imaging was performed on an LSM 710 confocal scanning microscope (Zeiss, Oberkochen, Germany), controlled by ZEN 2011 (Zeiss) software, equipped with a Plan-Apochromat 63× objective (NA 1.4, oil), 488, 561 and 633 nm laser lines, and continuous spectral detection. Images were acquired at 16-bit depth, 1024 × 1024 pixels, pixel size 0.132 µm, with 2× line averaging and a pixel dwell time of 6.3 µs, either as a single plane or as a Z-stack with 0.37 µm step size. In all channels, pinhole was set to 1 Airy unit. Emission light was collected sequentially, according to the emission spectra of the fluorophores used.
For the live-cell imaging, a temperature module was used and cells were placed in a heating insert (PeCon, Erbach Germany), which had been equilibrated to 37 °C. The medium used for imaging was Hibernate E medium containing 1% PS and 2% B27 Plus.

To label the endocytic pathway with dextran, cells were loaded for 16 h with 1 mg/mL dextran Alexa Fluor 488 10 000 MW, anionic (Thermo Fisher Scientific, D‐22910), washed, fixed, permeabilized and stained for intracellular tetherin as described above. To label endosomal/lysomal compartments containing the active acid hydrolase cathepsin B, live cells on coverslips were incubated for 5 min at 37°C with Magic Red MR‐(RR)₂ Cathepsin B substrate (1:2600 dilution) (ImmunoChemistry Technologies, 938) and then imaged on an incubated Zeiss LSM710 Confocal Microscope. To assess acidic organelles, cells were labelled with LysoSensor Yellow/Blue DND‐160 (Thermo Fisher Scientific, L‐7545), a ratiometric probe that produces blue fluorescence in a neutral environment but shifts to yellow fluorescence in more acidic compartments (pKa ≈ 4.2). Cells were incubated for 1 min at 37°C with LysoSensor and then imaged on an incubated Zeiss LSM710 Confocal Microscope. Cells were excited at 405 nm and images were taken at 455.5 ± 32.5 and 570.5 ± 70.5 nm of emission. Zen 2011 (Zeiss) and ImageJ software were used for the analysis and processing of confocal images. The graph shows the mean value with the standard error mean of the yellow/blue ratios obtained from three experimental repeats (20 cells spread across 4 confocal fields for each experimental condition).

Strains harboring P_GIS1-GIS1-mCHERRY were grown on YPGlcN agar at 30°C for 48 hrs or 168 hrs to examine the subcellular localization of Gis1. Calcofluor White (1 mg/mL) was used for cell wall staining before microscopic examination. Images were acquired using a Zeiss Axioplan 2 imaging system with the AxioCam MRm camera (Carl Zeiss Microscopy). The fluorescence intensity of Gis1-mCherry in yeast cells or hyphae was analyzed using software Zen 2011 (Carl Zeiss Microscopy).

Contrast and false colour images were optimized in Zen 2011 (Zeiss, Germany). Further image processing, including cutting and image mode conversion was done in Adobe Photoshop® CS4, figures were prepared in Adobe Illustrator® CS4 (Adobe Systems Software, Ireland).

Adherent cultures plated at 3 × 10⁴ cells per well in Lab-Tek™ chamber slides (Thermo Scientific®) were treated with the IC₅₀ of samples and controls (25 μg/mL vincristine Sigma® and 1X PBS vehicle) for 24 h. Then, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde (Sigma®) for 1 h at 37 °C. The cells were then washed with 1X PBS and stained. The morphology of the cells was determined using Hemacolor® rapid staining (Merck®), and the presence of apoptosis was assessed using the Apo-BrdU™ TUNEL assay kit with Alexa Fluor 488 (Invitrogen®), according to the manufacturer’s instructions. After applying both stains, the slides were treated with Vectashield®/DAPI mounting medium (Vector Laboratories®) or Entellan® resin (Merck®) according to the assay and analyzed by optical microscopy (BX41, Olympus®) or confocal microscopy (LSM 700, Zeiss®) using a 40X immersion lens with Image-Pro Plus (version 4.0, Media Cybernetics©) or ZEN 2011 (version 1.0, Zeiss®) software.

At indicated time-points (4, 24, 48, and 72 h) post-infection, organs (lung, liver, and kidney) were excised and fixed in 4% paraformaldehyde for 24 h. Tissues were processed, embedded in paraffin, and 4 µm vertical sections were fixed to glass slides. Tissue sections were stained with Hemotoxylin and Eosin (H&E), Gram or MPO to assess morphology, bacteria, or neutrophil infiltration, respectively. Microscopic examinations of tissues were performed by light microscopy with an Axiovert 40 CFL inverted microscope (Carl Zeiss, Thornwood, NY) and photographed with an AxioCam MrC digital camera using the Zen 2011 digital imaging software (Carl Zeiss).