The LysoTracker staining was performed on live cells as described previously (Chazotte, 2011 (link)). Stable LAMP1‐GFP RPE1 cell lines were generated with the pLVX‐EF1a‐LAMP‐1‐mGFP‐IRES‐Puromycin plasmid (Addgene #134868, a gift from David Andrews) (Schormann et al, 2020 (link)). Then, the cells were grown on coverslips in normal culture medium to ~80% confluence. The LysoTracker Red DND‐99 probe (Invitrogen L7528) was diluted to a working concentration of 50 nM in pre‐warmed (37°C) culture medium and applied to replace the normal culture medium. After the cells were incubated with the probe‐containing medium for 1 h under growth conditions, the loading medium was aspirated, and the cells were washed with fresh normal culture medium to remove any extra dye from the coverslips. The nucleus was stained with Hoechst 33342 (ImmunoChemistry #639) at a concentration of 0.5% v/v. The coverslips were then transferred onto slides and mounted with PBS. The slides were imaged immediately under a confocal microscope (Leica, TCS SP8). The number and size of LysoTracker‐positive puncta per cell were quantified. The colocalization of LysoTraker and LAMP1‐GFP was analyzed using the colocalization tool in Fiji software.