Cells were fixed for double immunofluorescence staining, which was performed as described previously (Hwang et al., 2019 (link)). First, the fixed cells were incubated with 1% bovine serum albumin and 0.02% Triton X-100 in phosphate-buffered saline (PBST–BSA) containing an anti-LC3 antibody (1:500, #2775, Cell Signaling, Danvers, MA, USA) overnight at 4°C, followed by Alexa Fluor® 488-conjugated anti-rabbit antibodies (1:500, Molecular Probes, Carlsbad, CA, USA) for 1 h at room temperature. Alternatively, the fixed cells were incubated with PBST-BSA containing anti-LAMP1 antibody (1:500, ab25630, Abcam, Cambridge, UK) overnight at 4°C, followed by Alexa Fluor® 594-conjugated anti-mouse antibodies (1:500, Molecular Probes) and Hoechst 33342 (10 µg/mL, Molecular Probes) for 1 h. The cells were washed three times with PBST for 10 min after each incubation step. After mounting, the cells were visualized under a confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).