Fig. 3.

Metformin disrupts interaction between hCAR and coactivators. Mammalian two-hybrid assays were performed in COS1 cells transfected with expression plasmids encoding VP16-AD/hCAR fusion proteins and GAL4-DBD/coactivator fusion proteins as indicated—(A) SRC1, and (B) GRIP-1—together with the reporter gene plasmid pG5luc for 16 hours. Cells were treated with vehicle control (0.1% DMSO), PB (1 mM), CITCO (1 µM), MET (1 mM), or their combination for another 24 hours before the determination of luciferase activities. Data represent the mean ± S.D. of three independent transfections (**P < 0.01). Coimmunoprecipitation assays were performed using COS7 cells transfected with expression plasmids encoding hCAR and coactivator proteins: (C) SRC1 and (D) GRIP1. The whole cell lysate was collected and incubated with DMSO (0.1%), MET (0.1, 1.0, 2.0 mM), PB (0.5, 1, 2 mM), CITCO (0.5, 1.0, 2.0 µM), or their combination overnight at 4°C. Proteins were immunoprecipitated and analyzed by Western blot analysis as outlined in Materials and Methods.