Fig 1.

AMPK activation suppresses LXR ligand-mediated induction of Srebp-1c promoter activity in McA-RH7777 cells. A, Diagram of plasmids containing the wild-type or mutant mouse Srebp-1c promoter linked to the firefly luciferase reporter gene. B, Luciferase reporter assay. On Day 0, McA-RH7777 cells were plated at density of 5 x 10⁴ cells per well in 24-well plates in Medium A supplemented with 10% FBS and incubated at 37°C in a 5% CO₂ incubator. On Day 1, cells were washed with 0.5 ml PBS and 0.5 ml fresh Medium A was added to each well before transfection. Cells were cotransfected with plasmid D and a control plasmid as described in Materials and Methods. Six hours after transfection, cells were washed with 0.5 ml PBS, switched to Medium A supplemented with 10% delipidated FBS and 0.5 mM AICAR or metformin or various amounts of T0901317, and incubated for 16 hours at 37°C and 5% CO₂. On Day 2, cells were washed with 0.5 ml PBS, and luciferase and beta-gal activities were measured as described in the Materials and Methods. Normalized firefly luciferase activity without treatment was arbitrarily set as 1.