Figure 6.

TPr-induced AMPK activation requires LKB1. Activation of AMPK by IBOP is LKB1-dependent (a). LKB1-deficient A549 cells were transfected for 24 hours with LKB1 wild-type (WT) or LKB1 mutants (S307A, S428A, and D194A). The plasmid encoding LacZ was used as a control. Transfected cells were stimulated with 1 µmol/L IBOP or with vehicle for 10 minutes. Data are means±SEM (n=3). ♣P<0.05, treated vs untreated cells. Adenoviral overexpression of LKB1 mutants abolishes IBOP-induced AMPK phosphorylation in VSMCs (b). VSMCs were infected with adenoviruses encoding LKB1 mutants D194A, S307A, and S428A, respectively. Cells were stimulated with 1 µmol/L IBOP for 10 minutes. Data are means±SEM (n=3). ♣P<0.05; ‡P<0.01, treated vs untreated cells. IBOP enhances the coimmunoprecipitation of LKB1 and AMPK in VSMCs but not in A549 cells transfected with LKB1 mutants (c). LKB1 was immunoprecipitated after IBOP treatment (1 µmol/L, 10 minutes) from VSMCs or A549 cells transfected with S307A, S428A, or D194A mutant LKB1 plasmids, and AMPK was then detected in Western blots. Data are means±SEM (n=3). ‡P<0.01, treated vs untreated cells. Effect of CaMKKβ on TPr-induced AMPK activation (d). VSMCs were preincubated for 6 hours with 1 µg/mL (2.6 µmol/L) STO-609 or transfected by CaMKKβ small interfering RNA to knockdown CaMKKβ expression; then the cells were treated with 1 µmol/L IBOP for 10 minutes. The blot is representative of 3 blots obtained from 3 independent experiments. Data are means±SEM (n=3). ♣P<0.05, treated vs untreated cells.