Figure 7
p62 is required for ESI-induced autophagy. (a) H1299 and A549 cells were treated with the indicated concentration of ESI for 24 h, and the p62 protein level was determined by western blot. (b) H1299 cells transfected with Flag-Keap1 plasmid were treated with ESI (1.6 μM) for 24 h. Immunoprecipitation was performed using an anti-Flag antibody or IgG as control, and immunublotting was carried out on the total cell lysates or immunoprecipitates using the indicated antibodies. (c) H1299 and A549 cells were transfected with pEGFP-p62 plasmid and pEGFP-N1 (vector control), and the autophagy markers including Beclin1, ATG3 and LC3 were determined by western blot. (d-f) H1299 cells overexpressing mCherry-LC3 were transfected with pEGFP-p62 plasmid and pEGFP-N1 (vector control) for 24 h, and the mCherry-LC3 punctate dots in cells were examined. Positive signals were defined if the cells have five or more mCherry-LC3 dots in the cytoplasm. (e and f) The number of mCherry-LC3 dots per cell (e) and the percentage of the cells with mCherry-LC3 dots (f) were counted under florescence microscope. (g and h) H1299 and A549 cells were transfected with anti-p62 siRNA (50 nM) or control siRNA together with indicated concentrations of ESI treatment, and expression levels of the autophagy markers (g) and cell viability (h) were analyzed. Note that knockdown of p62 attenuated ESI-induced autophagy and survival. All data were representative of three independent experiments. Bars, S.E.M.; *P<0.05, **P<0.01, ***P<0.001
