Figure 4.

64B directly binds to p300 and interferes with p300 recruitment to HIF-1 activated MET and CXCR4 genes. A, Chemical formula of 64B and inactive analog BW-HIF13. B, Dose-response of 64B and BW-HIF13 on hypoxia-induced luciferase expression in Mel290-V6R cells with a stably integrated HRE-luciferase reporter. (n = 3). C, Cellular thermal shift assays on 92.1 cell extracts treated with 64B or BW-HIF-13 (2 μM for 2 hours). Western blot (Left) of soluble fraction of individually heated cell extract aliquots (44–72°C) show 64B (but not BW-HIF-13) extends the thermal stability of p300. Quantification of p300 bands (Right). (n = 3). D, Chromatin immunoprecipitation (Chip) analysis shows hypoxia-induced binding of p300 on the CXCR4 and MET promoter regions is inhibited by 64B (2 μmol/L) in 92.1 cells. P300 recruitment to control gene DAPK1 was not affected. Data are representative of three independent experiments.