A9978

Male C57BL/6 mice were obtained from Chongqing Medical University. To induce hepatic carcinogenesis, 4-week-old C57BL/6 male mice received 75 mg/kg DEN (Sigma-Aldrich, N0258) in two divided doses and CCL4 (mixed with olive oil in a 1:4 ratio, 0.08 ml/10 g) twice a week until 20 weeks. After 8 weeks, fresh liver tissues were obtained through liver biopsy. Immunohistochemical staining was used to detect the level of AMPK phosphorylation. The same criteria with human tissue microarrays were adopted to distinguish the level of p-AMPK expression. In mice with p-AMPK high expression, dorsomorphin (Abcam, ab120843, 0.2 mg/kg d) was used to inhibit p-AMPK, or PBS as a control. In mice with p-AMPK low expression, metformin (Sigma, PHR1084, 250 mg/kg d) or AICAR (Sigma, A9978, 2.5 mg/kg d) were used to activate AMPK. For autophagy inhibition, mice received 50 mg/kg of chloroquine (Santa Cruz Biotechnology, sc-205629) via intraperitoneal injection once every 3 days along with a concurrent application of metformin. All mice were killed at 32 weeks. All animal experiments complied with the ARRIVE guidelines and was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.

HAP1 or SH-SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx-100 and Laemmli sample buffer. Along with protein ladder (Bio-Rad, 161–0373), the cell lysates or recombinant protein were loaded on a 4–20% Criterion gel (Bio-Rad, 3450032) for SDS-PAGE and transferred to PVDF membrane (Millipore, IPFL00010) using the semidry transfer method. In experiments with AICAR (Sigma, A9978), cells were exposed to the drug at 0.4 mM concentration for 72 hr before lysate preparation. Membranes were incubated in a blocking solution of TBS containing 5% nonfat milk and 0.05% Triton X-100. The membranes then incubated overnight with primary antibody solutions diluted in a buffer containing PBS with 3% BSA and 0.2% sodium azide. The following day, membranes were washed three times in TBS containing 0.05% Triton X-100 (TBST) and then incubated in secondary antibody (mouse HRP or rabbit HRP) diluted in blocking solution at room temperature for 30 min. The membranes were then rinsed in TBST three times and treated with Western Lightning Plus ECL reagent (PerkinElmer, NEL105001EA). Membranes were exposed to GE Healthcare Hyperfilm ECL (28906839) for visualization.