U-2 OS cells stably expressing HaloTag-LC3 (HT-LC3) were transfected for 24 h with pEGFP empty vector, pEGFP-VPS15-WT or pEGFP-VPS15-LS_403-410_AA or pEGFP-empty or TfR-4KA-GFP, and loaded with membrane-impermeable Alexa Fluor (AF)-660 HaloTag ligand (MIL), followed by membrane-permeable tetramethylrhodamine HaloTag ligand (MPL). Phagophores (PH), nascent autophagosomes (Au), mature autophagosomes and autolysosomes (MAu), total number of vesicles (Total) were quantified in GFP-positive cells. The histogram on top shows the number of HT-LC3 (% of total); the histogram on bottom shows total number of vesicles/cells. Data are mean ± SD (n= 3 three independent experiments [10 cells in each condition]). One-way ANOVA with Dunnett’s multiple comparisons test,*p< 0.05,**p< 0.01,***p< 0.001; ns, not significant.
