Pm045

Cells were seeded at 50% confluence. Next day, cells were treated with puromycin (5 µM) in full medium for 4 h. After the puromycin treatment, cells were washed twice with fresh medium and incubated in full medium for 4 h. During the second incubation, cells were treated with chemicals or starvation medium as indicated in each experiment. After the second incubation, cells were treated with 4% formaldehyde in PBS for 10 min at room temperature, and permeabilized with 0.3% Triton X-100 for 30 min at room temperature. Ubiquitinated protein aggregates in cells were stained using anti-poly-ubiquitin antibody (EMD-Millipore, 04-263; dilution 1:100) and/or anti-p62 antibody (MBL International, PM045; dilution 1:100). The concentrations of other used chemicals are the same as described in other assays. Images were taken using a Deltavision PersonelDV microscope as described above in Immunostaining and fluorescence microscopy section. For each analysis, 50 cells were counted across three independent experiments.

Slides of 4% PFA-fixed, paraffin-embedded sections (7 μm) were subjected to IHC analysis. After routine deparaffinization and rehydration, the slides were heat-induced at 100 °C in citrate sodium buffer (10 mM, pH 6.0) for 20 min to retrieve antigens. Subsequently, 3% H₂O₂ was used to suppress endogenous peroxidase activity, and blocking buffer (ab64226) was used to reduce nonspecific binding. Antibodies against αSMA (1:800; Abcam, ab124964), collagen I (1:100; Abcam, ab34710), SQSTM1/p62 (1:400; MBL International, PM045), and the negative control rabbit IgG were applied in the same manner for each sample. There was no specific staining detected in the negative controls.

Protein lysates (20–50 μg) of liver tissue and cells were separated in 12% sodium dodecyl sulfate gels and then were transferred onto polyvinylidene fluoride membranes. After blocking in 5% BSA in Tris-buffered saline and Tween-20, membranes were incubated with specific primary antibodies, including rabbit p-AMPK (1:1000; 2535S, Cell Signaling Technology, Danvers, MA, United States), rabbit AMPK (1:1000; 2532S, Cell Signaling Technology, Danvers, MA, United States), rabbit SIRT1 (1:1000; 2028S, CST), rabbit LC3B antibody (1:2000; L7543, Sigma-Aldrich, St. Louis, MO, United States), rabbit p62 (1:1000; PM045, MBL International, Des Plaines, IL, United States) and mouse β-actin antibody (1:3000; sc-376421, Santa Cruz Biotechnology, Santa Cruz, CA, United States) overnight at 4°C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated IgG. Blots were developed using ECL Detection Kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and then detected using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, United States). Protein bands were quantified using densitometry with Image J (NIH, Bethesda, MD, United States).