Ab56416

Retinal pigment epithelial‐1 cells used for protein analysis were plated at low density onto 6‐well cell plates (Eppendorf 30720113) in culture medium and treated for each experimental condition at 50% confluence. For whole‐cell protein analysis, cells were lysed with ice‐cold RIPA buffer containing protease and phosphatase inhibitors. Lysates were separated on a gradient gel (TGX, Bio‐Rad) and transferred to a PVDF membrane. Membranes were blocked with blocking buffer (LI‐COR Odyssey Blocking Buffer 927–40000) for 1 hr before probing with primary antibodies for p16 (Abcam ab108349), SQSTM1/p62 (Abcam ab56416), LC3B (Cell Signaling E5Q2K), and beta‐actin (Cell Signaling 8H10D10) in blocking buffer overnight at 4°C. Membranes were washed and probed with secondary antibodies (LI‐COR goat anti‐mouse IRDye800 and goat anti‐rabbit IRDye680) for 1 hr at room temperature and visualized using the LI‐COR Odyssey CLx Imaging System. Proteins were normalized to actin and quantified using ImageJ. Quantified results were tested for statistical significance in MATLAB using two‐way ANOVA and Bonferroni correction for multiple comparison analysis.

Protein expression was determined using the following antibodies in immunoblotting: p62 1:1000 (Abcam #ab56416), PINK1 1:1000 (Cell Signaling #9646), p53 (Abcam #ab17869), LC3 (Abgent # AM1800A), DRP1 1:500 (Abcam #AB56788), and MFN2 1:500 (Sigma #WH0009927M3). Left ventricular free-wall tissue was homogenized in protein lysis buffer (50 mM Tris–HCL pH 7.5, 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1 mM Phenylmethylsulfonyl fluoride, 1 tablet Roche Complete MINI protease inhibitor). Samples were centrifuged at 2.5 k RPM for 15 min, aliquoted, and stored at −80°C. A total of 15–25 μg protein was loaded into 4–15% TGX gels (#4561086, BioRad, Hercules, CA, United States) via SDS-PAGE, transferred onto Polyvinylidene difluoride (PVDF) membranes, and probed with mouse or rabbit antibodies specific for these proteins and subsequently probed with donkey anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher, Waltham, MA, United States). Blots were developed with SuperSignal West Pico (#34080, Thermo Fisher, Waltham, MA, United States), imaged with ChemiDoc 2000 (BioRad, Hercules, CA, United States), and quantified and analyzed using ImageLab (BioRad, Hercules, CA, United States) and ImageJ (US National Institutes of Health, Bethesda, MD, United States) softwares.

Nineteen clinical specimens of primary CRC with matching normal colonic mucosa and liver metastases were analyzed for expression of FASN (Cell Signaling, # 3180), p62 (Abcam, ab56416) and pAMPK (Cell Signaling, # 2535). Blind scoring performed by a pathologist used a semi-quantitative method. The extent of expression score was assessed on a scale of 0 to 3 (no positive cells = 0, <10% = 1, 10%–50% = 2, positive staining of >50% = 3); the intensity score was also measured on a scale of 0 to 3 (negative = 0, weak = 1, moderate = 2, strong = 3). Multiplication of the values for intensity and extent of expression provided a score for immunoreactivity.