Figure 1
ER stress inhibited autophagic flux by blocking autophagosome-lysosomal fusion. (a) Schematic showing the steps of maturation from phagophore to autolysosome in the autophagy machinery. MAP1LC3B-II (also known as LC3-II) and SQSTM1 (also known as p62) are bound to autophagosome membranes. Autolysosomes are generated by the fusion of autophagosomes with lysosomes expressing LAMP1. The inner membrane including MAP1LC3B-II and SQSTM1 is digested in the autolysosomes. (b) Western blots of HchEpC1b cells cultured with 500 ng/ml of brefeldin A (BFA) or 500 ng/ml of tunicamycin (TM) for 24 h, with or without 10 nM of bafilomycin A1 (Baf) treatment for 2 h at the end of culture, are shown as follows: BECN1, SQSTM1, MAP1LC3B (LC3), and TUBA. The graph shows the expression levels of MAP1LC3B-II (c) or SQSTM1 (d) in HchEpC1b cells, cultured with BFA or TM in the presence (white bars) or absence (black bars) of Baf. Expression was normalized to TUBA levels. (e) Representative panels showing the merged images of anti-LAMP1 staining (green), anti-MAP1LC3B staining (LC3, red) and nuclear staining (DAPI, blue) in HchEpC1b cells. The smaller images show the anti-LAMP1, anti-LC3, and the merged images of the area surrounded by white lines in the large images. For this experiment, the cells were cultured under serum free condition with 50 μM of chloroquine for 2 h at the end of culture, with DMSO (Cont), 500 ng/ml BFA or 500 ng/ml TM. As a negative control, HchEpC1b cells were cultured in RPMI1640 medium with 10% FBS without chloroquine. (f) The graph shows the average numbers of autophagosomes (Ap, black bars) or autolysosomes (Al, white bars) in HchEpC1b cells, which were cultured with DMSO (Cont), 500 ng/ml BFA, 500 ng/ml TM or 10% FBS. Data is expressed as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 20 μm.
