Polylysine coated glass slides

One milliliter of logarithmic cells were washed with PBS buffer, sonicated using 15 s pulses at 20% power and resuspended in 50 µL of ProtesoStat dye diluted 1:250 in ProteoStat assay buffer. Cells were incubated for 15 min at room temperature protected from light. 10 µL of each sample was deposited on poly-lysine-coated glass slides (Sigma-Aldrich). Cells were imaged using Leica TCS SP8 laser confocal microscope with ×63 1.4 NA or ×100 1.4 NA oil objectives. To visualize the nuclei, Hoechst (33342; ImmunoChimestry) was used. An argon laser with 488 excitation line was used for ProteoStat (emission wavelength, 500–600 nm), and a 405 UV laser was used to excite Hoechst fluorescence (emission wavelength, 415–450 nm). The results displayed are representative of three biological experiments.

Cells were grown to mid-log phase (OD₆₀₀ = 0.6) in SD medium supplemented with 2 mg/L adenine and the indicated concentrations of the chemical chaperones. One milliliter of cells was washed with PBS, sonicated for 2 min at 43 kHz (Ultrasonic Cleaner 0.6L 220V DG-1, MRC Labs, Israel) and resuspended in 50 µL of ProteoStat (prepared according to the manufacturer’s instructions) diluted 1:250 in PBS. Cells were incubated in the dark for 15 min at room temperature. 10 µL of each sample was deposited on polylysine-coated glass slides (Sigma-Aldrich). Cells were imaged using Leica TCS SP8 laser confocal microscope with ×100 1.4 NA oil objectives (Leica Microsystems, Wetzlar, Germany). Excitation and emission wavelengths of 488 nm and 500–600 nm, respectively. The results displayed are representative of three independent biological repeats.

Cells were seeded on polylysine-coated glass slides (Sigma) in 6-well plates, treated with the experimental drugs, incubated as necessary, stained with 70 ng/ml LysoTracker^® Red DND-99 (Invitrogen Life Technologies) for 30 min, washed with phosphate-buffered saline (PBS) at pH 7.2, fixed in 4% paraformaldehyde for 30 min, washed with PBS and permeabilized with 0.1% Triton-X-100 (Sigma) for 15 min, followed by overnight incubation with the LC3 primary antibody (1:1,000; MBL, Nagoya, Japan) and incubation with a secondary antibody (1:100; Kangcheng Biotech, Shanghai, China). A laser-scanning microscope (Olympus BX60; Olympus, Tokyo, Japan) was used to capture images.