Digitonin

On the day before treatment, cells were grown on glass coverslips (Marienfeld). After treatment, cells were fixed with 4% formaldehyde-PBS for 30 min on ice and quenched with 50 mM NH₄Cl for 15 min. For immunofluorescence labeling, cells were then permeabilized with 0.2% Triton X-100-PBS for 15 min, or 50 µg/ml digitonin (Roth, #4005) for 5 min according to the antibody manufacturers. Samples were blocked with 3% BSA (Roth, #8076)-PBS for 30 min and incubated with primary antibodies for 1–2 h. After washing and 30 min of secondary antibody incubation, samples were again washed three times with PBS. Cells were embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, #P36980), including DAPI. Imaging was performed with a Zeiss Axio Observer 7 fluorescence microscope (Zeiss, Köln, Germany) with a Plan Apochromat 40x/1.4 oil objective (Zeiss, Köln, Germany). Quantification of images was performed with ImageJ. For that, signals and nuclei were counted per image and a signal-to-nuclei ratio was calculated. Macros for the quantifications are provided in Supplementary Methods.

In all, 5 × 10⁴ cells were plated on glass coverslips one day prior to treatment. After treatment, the cells were fixed on ice using 4% paraformaldehyde-FBS for 15 min, quenched with 50 mM NH₄Cl for 15 min, and permeabilized with 50 µg/ml digitonin (Roth, #4005) for 5 min. Subsequently, the cells stably expressing mRFP-EGFP-rLC3 were directly stained with DAPI. The other cells were blocked with 3% bovine serum albumin (Roth, #8076)-PBS for 30 min, and then incubated with indicated primary antibodies and corresponding secondary antibodies for 1 h each. Cells were embedded in ProLong Glass Antifade Mountant (Thermo Fisher Scientific, #P36980) containing DAPI. For BODIPY 581/591 C11 staining, 2.4 × 10⁴ of 253J or T24 cells per well were seeded on ibidi µ-Slide 8 Well (ibidi #80826). The following day, cells were treated with Fin56 (2 µM) or DMSO for 4 h. After treatment, cells were incubated with 2 μM BODIPY 581/591 C11 (Thermo Fisher Scientific, #D3861) for 30 min. Subsequently, cells were washed twice with PBS and subjected to microscopy. The Zeiss Axio Observer 7 fluorescence microscope (Zeiss, Köln, Germany) with a Plan Apochromat ×40/1.4 oil-objective (Zeiss, Köln, Germany) was used for imaging. The images were quantified with ImageJ, and the macros used for quantification are listed in the supplementary methods.

Membranes prepared from insect cells (1 mg of total protein) were resuspended in 0.4 ml lysis buffer L1 (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 2% (w/v) digitonin (Carl Roth, Karlsruhe), 1.5 mM phenylmethylsulfonylfluoride, 3 mM benzamidine). After solubilization for 60 min on ice, non-solubilized proteins were removed by centrifugation at 100,000× g for 30 min at 4°C. The supernatant was incubated with M-280 sheep anti-mouse IgG Dynabeads (Dynal Biotech, Hamburg), which had been pre-loaded with antibodies. Beads were washed three times with 1 ml of washing buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.2% (w/v) digitonin). Proteins were eluted in 1× SDS sample buffer (2% SDS, 50 mM Tris/HCl, pH 8.0, 200 mM DTT, 10% glycerol, 0.05% bromophenol blue) for 3 min at 65°C. Samples were denatured for 20 min at 65°C and separated by SDS-PAGE (12% or 6%). After electro transfer onto nitrocellulose membranes, proteins were detected with specific antibodies as indicated. Horseradish peroxidase (HP) conjugated secondary antibodies were detected with Lumi-Imager F1 (Roche). One representative blot out of three independent experiments is shown.