Overexpression of PDK1 increases MMP-2 activity and MT1-MMP. A, Comma/Vector and Comma/PDK1 cells were grown and conditioned medium was concentrated and assayed for MMP activity by zymography in the present or absence of 1 mM APMA as described in Methods. Protein markers are indicated by their mass in kDa. B, Concentrates of conditioned medium described in A containing 2 μg protein were separated under non-reducing conditions by SDS-PAGE in 10% polyacrylamide gels, and blotted onto nitrocellulose and analyzed with an anti-MMP-2 antibody. C, Comma/Vector (Vector) and Comma/PDK1 (PDK1) cells were incubated for 24 hr in the presence and absence of 1 μM lactacystin, followed by incubation in serum-free medium for 24 hr. Samples were assayed by zymography as in A. D, Cells were transfected with 'WT' or 'D9' MMP-2 promoter plasmids as described in Methods. Luciferase activity was measured after 24 hr and is expressed as the -fold change relative to control cells. Luciferase activity (light units) in control cells with the 'WT' and 'D9' plasmids was 2,970 and 6,520, respectively. E, Comma/Vector (Vector) and Comma/PDK1 (PDK1) cells were treated with or without lactacystin as in B and cell lysates analyzed by western blotting for levels of MT1-MMP.