Bafilomycin

Wild‐type, ATG5^−/−, and STG‐p62 Hap1 cells were starved for 2 h in Earle's balanced salt solution (EBSS, Sigma) with or without addition of 400 nM Bafilomycin (Santa Cruz Biotechnology) to block lysosomal degradation, lysed, and subjected to Western blotting. For blotting against LC3B, the lysates were heated up only to 60°C for 10 min after the addition of Laemmli sample buffer.

Cells were seeded onto 8-well glass slides at a density of 1.5 × 10⁴ cells/well and incubated overnight in the presence of 0.1 µg/ml (isoform 1) or 1 µg/ml (EV and isoform 2) doxycycline. 24 h post-induction, the culture medium was replaced and cells were treated with either 100 nM Bafilomycin (Santa Cruz Biotechnology) or vehicle control (DMSO) for 4 h. Subsequently, cells were fixed with 4% formaldehyde (Thermo Scientific) and incubated with blocking buffer (5% normal goat serum/0.3% Triton X-100 in PBS) for 1 h at room temperature. Following staining with primary antibodies diluted in PBS containing 1% BSA and 0.3% Triton X-100 overnight at 4 °C, cells were incubated with Alexa Fluor 546 or 555 anti-mouse or anti-rabbit IgG secondary antibodies (Life Technologies) for 1 h at room temperature and counterstained with DAPI (Invitrogen, Carlsbad, CA). Finally, slides were mounted in Fluoroshield (Sigma-Aldrich) and images were acquired on a confocal microscope (Olympus FLV1200). Representative images of at least three independent experiments are shown. LC3B puncta were quantified using CellProfiler software and the number of puncta was normalized to the number of nuclei.

For siRNA treatment cells were seeded in six-well plates (110,000 cells/well). Cells to be analyzed by immunocytochemistry were seeded on coverslips. Twenty-four hours after seeding cells were transfected with siRNA (20 nM final concentration) using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) in OptiMEM medium. Forty-eight hours after transfection cells were left untreated or treated as specified in the figure legends and either harvested for western blot analysis or fixed for immunofluorescence analysis. When cell treatment with drugs was performed the following conditions were used: bafilomycin (Santa Cruz Biotech. – 400 nM for 2 h); wortmannin (Sigma – 1 µM for 2 or 3 h, as specified in the figure legend); puromycin (ThermoFisher – 5 µg/ml for 3 h); MG132 (Boston Biochem – 10 µM for 3 h); 1-NAA (Sigma – 500 µM – 1 mM for 3 or 12 h, as specified in the figure legend); doxycycline (50 ng/ml for 12 h).