A Novel Function of Novobiocin: Disrupting the Interaction of HIF 1α and p300/CBP through Direct Binding to the HIF1α C-Terminal Activation Domain
Figure 5
Novobiocin represses the transcriptional activity of HIF1α CTAD and down regulates HIF1α target genes.
A. Novobiocin inhibits the activation of GAL4-dependent luciferase reporter in 293T cells. 293T cells were co-transfected with luciferase reporter carrying GAL4-Luc and a Renilla luciferase plasmid (HIF1α DBD and HIF1α CTAD). After 24 hours transfection, cells were treated with 50, 100, and 200 µM novobiocin with or without CoCl2. After 48 hours transfection, dual luciferase activities were measured and firefly values were normalized with Renilla values. B. 293T cells were co-transfected with GAL4-Luc and VP16 plasmid. After 24 hours transfection, cells were treated with 50 and 100 µM novobiocin. C. Novobiocin down-regulates HIF1α target genes. A549 cells were cultured with or without 200 µM novobiocin for 24 hours in the presence or absence of CoCl2 (150 µM). Selective HIF1α target or non-target gene expressions were measured by RT-PCR (left panel). Western blot analysis was performed with indicated antibodies (right panel). D. Novobiocin affects mTOR gene expression in MCF-7 cells. MCF-7 cells were treated with 200 µM novobiocin in the presence or absence of CoCl2 (150 µM). Indicated gene expressions including Akt1and mTOR were measured by RT-PCR (upper panel). HIF1α protein is shown in the lower panel. E. mRNA was quantified by densitometry using Quantity One software (BioRad) (right panel). Error bars represent the standard error of the mean of 2∼3 independent experiments. The Student’s t-test was performed to compare difference between with or without novobiocin treatment (significance: *p<0.05 and **p<0.01).