Lipopolysaccharide Stimulates p62-Dependent Autophagy-Like Aggregate Clearance in Hepatocytes
Figure 3
LPS-induced autophagy is dependent on p62. (a) WT hepatocytes were treated with LPS (100 ng/mL) from 2 h to 24 h. p62 protein levels were determined by western blot analysis (left upper image) and mRNA levels were quantified using RT-PCR (right upper image). Data represents mean ± SEM; *P ≤ 0.05 versus control (time 0) level. Whole cell lysates from LPS-treated WT hepatocytes were also immunoprecipitated with anti-ubiquitin antibody and then immunoblotted for p62 and polyubiquitin (lower image). (b) Immunoblots for p62 and LC3II in whole cell lysates from WT hepatocytes pretreated with either control siRNA or siRNA targeting p62 followed by LPS stimulation (100 ng/mL) for up to 24 h. (c) Confocal immunofluorescence of GFP-LC3 puncta with quantification (right hand graph) in WT hepatocytes pretreated with control or p62 siRNA followed by LPS stimulation (100 ng/mL) for up to 16 h. (d) Confocal immunofluorescence of GFP-LC3 (green) with p62 (red) showing colocalization (merge = yellow) in WT hepatocytes pretreated with control or p62 siRNA prior to stimulation with LPS (100 ng/mL) for up to 4 h. Arrows indicate p62/LC3 aggregates. Data represents mean ± SEM; versus control siRNA LPS 4 h. Images representative of at least 3 separate experiments. Confocal images of 60x magnification with 1.5x zoom. LC3 puncta quantified for ≥100 cells and data normalized by number of nuclei.
