(A) Saos-2 cells were transiently transfected with 5 μg PUMA luciferase plasmid and 200 ng each of the indicated expression constructs. After 72 hours, cells were assayed for luciferase activity, and the data were normalized against transfected β-gal activity. Values represent fold activation relative to the activity of GFP alone, which was assigned as 1. (B) Saos-2 cells were infected as indicated with adenoviruses expressing tr105, 37AA, ΔN-p73 adenovirus, or control empty adenovirus. At the indicated time points, RNA was isolated from the cells and subjected to RT-PCR for PUMA and GAPDH. (C) At the same time cells were harvested and assessed for changes in cell death by flow cytometry. (D) Cells were infected with the indicated adenoviruses for 48 hours. The mRNA levels for PUMA, DR5, Bax, and p21 were determined by qPCR. Samples were normalized to the levels of 18S ribosomal RNA. (E) Schematic of the mode of action of 37AA. Introduction of 37AA into p53-null cells sequesters iASPP and thereby derepresses p73. TA-p73 subsequently activates apoptotic target genes such as PUMA to bring about programmed cell death. In line with this model, cell death from 37AA can be inhibited by decreasing p73 activity or increasing iASPP.