Digitonin

Autophagy in HeLa 229 cells was induced by nutrient-starvation or rapamycin treatment. For nutrient-starvation, HeLa 229 cells were washed and incubated with Krebs Ringer bicarbonate buffer pH 7.6 (KRB; 118.5 mM NaCl, 4.47 mM KCl, 1.18 mM KH₂PO₄, 23.4 mM NaHCO₃, 6 mM glucose, 2.5 mM CaCl₂, 1.18 mM MgSO₄, and 6 mg/l phenol red) for 0–6 h. For rapamycin treatment, cells were incubated with 1 µM rapamycin [stock 1 mM in dimethyl sulfoxide (DMSO), Sigma Aldrich, St. Louis, MO] in MEM for 4 h. Lysosomal protease inhibitors, 10 µg/ml E64d (Peptide Institute, Inc., Osaka, Japan) and 10 µg/ml pepstatin A (Peptide Institute, Inc.) were used to inhibit the lysosomal turnover. For immunostaining, the cells were fixed with 4% paraformaldehyde (Wako), washed with PBS, and lysed with 50 µg/ml digitonin (Wako). After quenching in 50 mM NH₄Cl, the cells were blocked in 2% (w/v) bovine serum albumin, 5% (v/v) normal goat serum in 20 mM Tris-HCl, 150 mM NaCl. Lysosomes were immunostained with anti-lysosomal-associated membrane protein 1 (LAMP1) antibody (Sigma Aldrich) and rhodamine-conjugated anti-rabbit immunoglobulin G (IgG) (MP Biomedicals, Irvine, CA), whereas autophagosomes were immunostained with anti-LC3 antibody (Sigma Aldrich) and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (MP Biomedicals). Fluorescence signal was observed under confocal microscope.

Cells seeded in 384-well black plates with clear bottoms (Cell Carrier Ultra; PerkinElmer) were treated with compounds for 24 h, then cells were fixed with 4% paraformaldehyde and permeabilized with 50 μg/mL digitonin (Wako Pure Chemical Co.) in PBS. After washing with PBS, cells were incubated with 10% goat serum (Thermo Fisher Scientific) for 1 h. After blocking, cells were incubated with anti-LC3 for 1 h, followed by incubation with an anti-rabbit secondary antibody conjugated to Alexa Fluor-488 (Thermo Fisher Scientific) and Hoechst-33258 for 1 h. After washing with PBS, images were captured by CV1000 (Yokogawa Electric Corp., Tokyo, Japan) using a 40× objective.

HeLa cells were fixed with 4% paraformaldehyde (Wako), permeabilized with 50 μg/ml digitonin (Wako), and incubated with primary antibodies followed by 1:2000 secondary antibodies (Alexa Fluor 488- or 568-conjugated goat anti-mouse or anti-rabbit IgG antibody; Invitrogen). Microscopy images were captured on a laser-scanning microscope (LSM710 or LSM780; Carl Zeiss), and image brightness was adjusted with Photoshop software (Adobe).

Cells were lysed in PBS containing 1% Digitonin (WAKO), PMSF and IAA. Supernatants were pre-cleared with protein A sepharose, immunoprecipitated with anti-tapasin PaSta-1 antibody (kind gift from Peter Cresswell) and complexes were recovered with protein A. Beads were washed in 0.1% Digitonin and eluted with Laemmli sample buffer. Eluted proteins and samples of lysate, prior to immunoprecipitation were separated by SDS-PAGE, transferred by Western blot and detected by chemilluminescence using Fluor-S Multimager. Anti-human TAP1, anti-HLA-B (N-20), HRP-conjugated, anti-rabbit light chain and anti-goat antibodies were used.

Sub-confluent 7WD10 cells in 35-mm glass-bottom dishes were treated with mouse monoclonal anti-human APP antibody 6E10 (1:250 dilution) and biotinylated LME-tet (50 µM) on ice for 30 min. After washing, cells were incubated at 37 °C for the indicated times, fixed with 4% paraformaldehyde (PFA), and permeabilized with 0.2% TritonX-100. APP was detected using Alexa Fluor 546-conjugated goat anti-mouse IgG antibody. LME-tet was detected using Alexa Fluor 488-conjugated streptavidin. Early endosome antigen 1 (EEA1) was detected using rabbit polyclonal anti-EEA1 antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. Lysosome-associated membrane glycoprotein 1 (LAMP1) was detected using rabbit polyclonal anti-LAMP1 antibody, followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody. For detection of the intracellular acidic compartment, cells were treated with 75 nM Lysotracker DND-99 (Molecular Probes, Eugene, OR, USA) for 1 h, followed by PFA fixation and permeabilization with 500 µg/ml digitonin (Wako Chemicals). Fluorescent images were analyzed using LSM700 laser scanning confocal microscopy (Zeiss, Oberkochen, Germany) and Image J software (NIH).