PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P
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