FIG. 2.
Depsipeptide induces p21Waf1/Cip1 expression through both p53 and Sp1. A wild-type p21 promoter luciferase fusion plasmid (pWWP-Luc) and three plasmids with mutated p21 promoters (pWWP-p53 mut1-Luc, pWWP-p53 mut2-Luc, and pWWP-Sp1-3-mut-Luc), were transfected into A549 cells. At 24 h after transfection, depsipeptide (0.1 μM) or TSA (2 μM) was added onto the cells for 6 h. Eighteen hours after depsipeptide or TSA treatment, cells were harvested and relative luciferase activity was measured (A). The luciferase activity was normalized for the amount of protein in the cell lysate. All of the luciferase experiments were carried out at least three times in triplicate. Plasmids with wild-type p53 were transfected into H1299 cells, and depsipeptide (0.1 μM) was added into cells for 6 h. Eighteen hours after depsipeptide treatment, cells were harvested and protein was extracted for Western immunoblot analysis with anti-p53 (P240) (B) and anti-p21 (C). In addition, H1299 cells were treated as for panel B, and RNA was extracted for RT-PCR assay to detect changes in p21 mRNA (D). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a loading control for the RT-PCR.