Fig 1: CPUL1 interferes with lysosomal function and fusion with autophagosomes. (A,B) The cells were treated with CPUL1 (8 μM) and 3-MA (2 mM) or CQ (20 μM) for 48 h, immunolabeling with the p62 and LC3 antibodies. DAPI (blue) was used to stain the nuclei, and the fluorescent images were observed with laser scanning confocal microscopy and processed using the ZEN imaging software. Scale bar = 20 μm. (C) The cells were treated with CPUL1 (8 μM) and 3-MA (2 mM) or CQ (20 μM) for 48 h, and the level of LC3-II/I and p62 was detected by Western blotting. (D) The cells were treated with CPUL1 (8 μM) and CQ (20 μM) for 48 h, and the colocalization of LC3 (667 nm red) and LAMP1 (570 nm orange) was assessed. DAPI (blue) was used to stain the nuclei, and the cells were photographed under a fluorescence microscope. Scale bar = 20 μm. (E) Cells were treated with CPUL1 (8 μM) and CQ (20 μM) for 48 h and stained with Lyso-Tracker Red for 60 min, Hoechst 33342 (blue) was used to stain the nuclei and visualized under a fluorescence microscope. Scale bar = 20 μm. (F) Cells were treated with CPUL1 (8 μM) for 6 h and 48 h, and the levels of RAb7, LAMP1, and LAMP2 were detected by Western blotting.