Immunocytochemistry and LysoTracker staining

N2A and HEK293FT cells were cultured on round coverslips in a 24-well dish. For immunostaining of HEK293FT cells, cells were fixed in 10% formalin/phosphate buffered saline (PBS) solution for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X−100/PBS for 10 min at RT. For co-staining of N2A cells with LysoTracker, cells were first incubated with LysoTracker Red DND-99 (1/10,000, L7528, Thermo Fisher Scientific) for 1 h at 37 °C before fixation, and permeabilization step was omitted. After washing twice with PBS and blocking with 3% bovine serum albumin (BSA)/PBT (PBS + 0.1% Tween-20) for 1 h at RT, cells were incubated overnight at 4 °C with rat anti-HA (1/200, 11867423001, Roche) for both HEK293FT and N2A cells, and also rabbit anti-ATP6V1A (1/500, ab137574, Abcam) and mouse anti-Lamp2 (1/200, ABL-93, DSHB) for HEK293FT cells as primary antibodies. After washing three times with PBS, cells were incubated for 1 h at RT with Alexa Fluor 488 donkey anti-rat IgG (for both HEK293FT and N2A cells), and with Alexa Fluor 546 donkey anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (for HEK293FT cells, 1/500 dilution, Thermo Fisher Scientific) as secondary antibodies. After washing three times with PBS, cells were stained with the nuclear marker DAPI and mounted with VECTASHIELD Hard Set Mounting Media (H−1400, Vector). Experiments were repeated twice. Fluorescent images of each cell line were taken from five independent regions using the Leica TSC SP8 confocal microscopy (Leica Microsystems) with identical condition, through a 63× objective lens with or without 4× digital zoom. For LysoTracker stain, the fluorescent intensity of five images of each cell line was measured using Image J software. Ratios to average intensity of untransfected control cells were calculated.