Filipin

Cells were seeded on coverslips, transfected 18 h later and treated as indicated. Twenty-four hours later, cells were fixed in 3.7% formaldehyde for 10 min and permeabilized in ice-cold methanol for 2 min. Staining was performed with the antibodies mentioned above and 4,6-diamidino-2-phenylindole (Invitrogen) to stain DNA or Filipin (Cayman Chemicals) to detect cholesterol. Images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) at × 63 magnification. For live-cell imaging, the microscope was equipped with a climate control chamber and cells were imaged for 8 min with ∼5 frames per minute. Images were quantified using Image J plugin Jacob for Mander's coefficient calculations or FIJI's Trackmate plugin for live-cell imaging and processed using Adobe Photoshop and Illustrator.

Cell were seeded on cover slips for different treatment. Cover slips were fixed in 4% PFA for 15 min and permeabilized with 0.3% Triton X-100 in PBS for another 15 min at room temperature. Then cells were incubated with Rhodamine Phalloidin (ABclonal, RM02835, 1:200) for 30 min or 50 µg/mL filipin (Cayman, 70440) for 1.5 h. Nuclei were counterstained with DAPI for 5 min (for F-actin staining). Images of the stained samples were captured with Leica TCS-SP8 and Leica TCS-SP8 DIVE.

Lipid distribution in the retina was assessed as described [41 (link), 63 (link), 64 (link)] by stains with filipin (Cayman Chemical, Ann Arbor, MI, USA, #70440) for UC and Bodipy 493/503 (ThermoFisher Scientific, Inc., Waltham, MA, USA, D3922) for UC, EC, triacylglycerides, and free fatty acid.

The presence and distribution of phosphatidylserine and cholesterol residues on GBM cells plasma membrane were analyzed with a fluorescence microscope Eclipse 80i (Nikon, Kawasaki, Kanagawa Prefecture, Japan). Phosphatidylserine residues were labelled for 10 min at 37 °C with 0.5 mg/mL of FITC-conjugated annexin-V (#A9210; Sigma-Aldrich, St. Louis, MO, USA) in the complete culture medium of cells grown on coverslips (1 × 10⁵ cells/13 mm diameter coverslip) after two extensive rinsings with 0.2M of PBS (pH 7.4).
Cholesterol was labeled for 2 h at 37 °C with a 0.05-mg filipin (#70440; Cayman Chemical, Milan, Italy)/mL of PBS (0.2 M), pH 7.4, in complete culture medium of cells grown on coverslips (1 × 10⁵ cells/13 mm diameter coverslip) after two extensive rinsing with 0.2M of PBS (pH 7.4).
Before fluorescence microscopy, observation slides were extensively rinsed with 0.2M of PBS (pH 7.4) and glycerine mounted medium.

To test the sterol binding ability, MoAtg9-GFP, MoAtg9^6A-GFP, MoAtg9^6D-GFP, MoAtg9^5A-GFP, MoAtg9^5D-GFP, and GFP (negative control) were purified using anti-GFP beads and incubated with 10 µM ergosterols (Solarbio, SE8200) for 45 min and then stained with 50 µg/mL filipin (Cayman Chemical, 70440) for 4 h and observed by fluorescence microscopy with a UV filter. Before imaging, the beads were washed three times with PBS and then viewed under a UV filter set (50 (link)).

HeLa cells were plated in a 6-well plate (2 × 10⁵ cells/well) and allowed to adhere overnight. Cells were infected with either mCherry-expressing WT or Δstmp1 mutant bacteria for 2 h, washed extensively with PBS, and incubated in growth media. At 2 dpi, the infected cells were replated onto coverslips in a 24-well plate (5 × 10⁴ cells per well). The next day cells were treated with dimethyl sulfoxide (DMSO) control or U18666A at 5 μM for 4 h. After the drug treatment, the cells were fixed with 2.5% PFA on ice for 15 min and incubated with 1:100 filipin (5 mg/mL stock in DMSO; Cayman Chemicals, Ann Arbor, MI) in PBS with 1% BSA for 1 h. Following washing with PBS, coverslips were mounted with ProLong gold and visualized on a Leica inverted DMI6000B microscope with a 63× oil immersion objective. Images were captured under identical capture settings and processed identically using ImageJ (30 (link)). At least 30 CCVs were measured per condition for each of the three independent experiments.

Three dyes were used as described (31 , 32 (link)). Fluorescent antibiotic filipin (catalog no.: 70440; Cayman Chemical, Ann Arbor, MI) visualized unesterified and esterified (after additional processing) cholesterol (UC and EC, respectively). Fluorescent compound Bodipy 493/503 (D3922; Thermo Fisher Scientific, Inc, Waltham, MA) stained UC, EC, triacylglycerides, and free fatty acids (33 (link)). Oil Red O (catalog no.: KTORO; StatLab, McKinney, TX) labeled EC, triacylglycerides, free fatty acids, and retinyl esters (6 (link), 34 (link)).