Filipin

Various inhibitors were used to study the transport mechanisms of Aβ and its isoforms. The contribution of RAGE to the transport of Aβ across the endothelial monolayer was assessed using the antagonist of this receptor FPS-ZM1 (Sigma) at a concentration of 20 μM (to obtain a stock solution, FPS-ZM1 was dissolved in DMSO to a concentration of 305 mM). To study the caveolin-dependent transport of Aβ isoforms, the inhibitor filipin (Sigma) was used at a concentration of 3 μg/ml (to obtain a stock solution, filipin was dissolved in DMSO to a concentration of 5 mg/ml). An equivalent amount of DMSO was added to the control samples. The contribution of clathrin-dependent endocytosis was assessed using chlorpromazine (Merck) at a concentration of 5 μg/ml (to obtain a stock solution, chlorpromazine was dissolved in DMEM). These concentrations were selected based on literature data and tested for toxicity to bEnd.3 cells using MTT (filipin) and WST (FPS-ZM1 and chlorpromazine) assays according to the manufacturer's protocol (Supplementary Figure 2). Before experiments, cells were preincubated with inhibitors added to the upper transwell compartment for 1 h, after which they were filled with solutions containing the inhibitor and 1 μM Aβ, and samples were taken from the lower compartment after 2, 6 and 24 h.

Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 30 min. For filipin staining cells on coverslips were stained with 25 μg/ml filipin (Sigma) in PBS in the dark for 2 h at RT, washed with PBS, mounted in Fluoromount G (EMS), and imaged. In some experiments nuclear staining was performed using SYTOX Green or 7-AAD from SelectFX Nuclear Labeling Kit (Invitrogen). For PFO∗ staining cells were permeabilized with 0.1% TX-100 in Cell Staining Buffer (BioLegend) for 30 min and then blocked for 1 h in Cell Staining Buffer. iFluor-647-PFO∗ (Codex Bio solutions and AAT Bioquest, custom order) was then added in Cell Staining Buffer for 1 h. Cells were washed three times with PBS and stained with 1:10,000 HCS CellMask Green (ThermoFisher) and 1:5000 Hoechst 33342 (ThermoFisher) in PBS for 15 min. Cells were washed with PBS and imaged.

For filipin staining, cells were fixed with 4% paraformaldehyde and quenched with 50 mM NH₄Cl followed by incubation with 0.05% filipin (Sigma) in PBS. After washing, the cells were mounted with Mowiol and imaged with a Nikon Eclipse Ti-E inverted wide field microscope equipped with a Nikon motorized stage and an Andor iXon + 885 EMCCD camera with ×40/0.75 NA air objective. The microscope was controlled with NIS-Elements Advanced Research 4.2 software (Nikon).
Late endosomal filipin fluorescence in LIMP-2-mCherry-expressing cells was quantified by ImageJ. The outlines of LIMP-2-positive and LIMP-2-negative LEs (within the same cell) were traced manually. After subtracting background fluorescence, the mean filipin intensity within individual organelles was measured.
Cells in Fig. 2 and S2, except cells in S2F, were visualized using a Zeiss LSM 800 confocal microscope (Zeiss) equipped with an Airyscan detector, and a ×63 oil immersion objective (1.4 NA; Zeiss). Images were acquired and processed with ZEN Blue software (Zeiss). Cells quantified in Fig. 2i and presented in S2F were visualized with a Zeiss ×63 1.4 NA oil-immersion lens using a spinning-disk confocal microscope (Quorum) equipped with a back-thinned EM-CCD camera (C9100-13, Hamamatsu); images were acquired by and processed with Volocity software (Perkin-Elmer).

Cells were grown at 80% to 90% confluency on 18-mm sterile cover glasses in 12-well plates and fixed with 4% paraformaldehyde (Alfa Aesar) at room temperature (RT) for 20 min. Then they were washed thrice with PBS, permeabilized, and blocked in a solution of 10% FBS and 0.01% Triton X-100 in PBS (PBSST) for 1 hour. To detect PrP^Sc-specific signals, cells were treated with 6 M guanidinium chloride (GdnCl) for 7 min after blocking and probed with anti-PrP (4H11) antibody as described (85 (link)). Otherwise, cells were incubated with primary antibodies (in PBSST) at their respective working concentrations for 1 hour at RT. The cells were then washed thrice in PBS and were incubated in appropriate anti-rabbit/mouse–Alexa dye coupled secondary antibodies (in PBSST) at their respective dilutions for 1 hour. Samples were washed again thrice with PBS and mounted on glass slides. Cells were imaged with a 63× oil immersion objective lens using a Zeiss LSM 700 confocal microscope. Three-dimensional z-stacks were acquired with a step size of 0.3 μm.
For filipin staining, following the fixation and the blocking steps cells were treated with 125 μg/ml filipin (Sigma) in PBSST for 2 h and imaged with a 63× oil immersion objective lens using a Leica DMI3000 B fluorescence microscope.

1.8 × 10⁴ A549 cells were cultured in 24-well plates with complete DMEM. After 72 h, A549 cells were maintained overnight in FBS-free DMEM (FBS starvation). Next, A549 cells were incubated with 2.0 × 10⁶H. capsulatum yeasts [multiplicity of infection (MOI) of 8 yeasts per A549 cell] for 5, 16, or 24 h. After incubation with H. capsulatum, culture supernatants were collected and centrifuged at 1300 × g to remove fungi. IL-6, IL-8, and IL-10 levels in these supernatants were determined using DuoSet^® ELISA Kits (R&D Systems), according to manufacturer’s instructions.
In some experiments, after FBS starvation, A549 cells were incubated for 2 h in FBS-free DMEM containing 0.1, 1, or 10 μM PP2 (an inhibitor of SFK activation, Calbiochem, USA), 1 μg/ml filipin (a cholesterol-binding compound that disrupts membrane rafts, Sigma, USA), or 0.025% or 0.05% DMSO (DMSO concentrations used as vehicle for PP2 and filipin, respectively). And then, H. capsulatum yeasts were added to the cultures and incubated for 16 h. IL-6 and IL-8 levels in these culture supernatants were determined as described above.