FIG 9.

p53 requirement for activation of Zp transcription by HIF-1α/β maps to the HRE. (A) Reporter assays showing that the HRE is the primary cis-acting element required for Zp activation by MLN-4924. AGS cells maintained in 24-well plates were transfected with 0.20 μg of DNA/well of the indicated luciferase reporter plasmid variants of pZp-Luc. Twenty-four hours later, 5 μM MLN-4924 or DMSO (the drug diluent) was added, and incubation was continued for an additional 24 h prior to harvesting and assaying for luciferase activity. Data were normalized to the values obtained with the vehicle control; they are presented as means ± SEMs from assays performed in triplicate on two independent occasions relative to the values obtained with pTATA-Luc. (B) Immunoblots showing that incubation of AGS cells with 5 μM MLN-4924 for 24 h leads to high-level accumulation of phosphorylated p53 as well as HIF-1α in these cells.