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Figure 1 | BMC Molecular Biology

Figure 1

From: The p53-inhibitor Pifithrin-α inhibits Firefly Luciferase activity in vivo and in vitro

Figure 1

PFT-α inhibits both p53-dependent and -independent firefly luciferase reporters. (A) PFT-α inhibits p53-dependent luciferase reporter plasmids. U-2 OS cells were transfected with 1.5 μg of the indicated reporters and treated with 20 μM PFT-α for 24 hours. Cells were harvested and luciferase activity was measured and normalised for total protein. Results are expressed as fold repression above untreated transfected cells, and represent mean plus standard deviation of a minimum of 3 independent experiments. (B) PFT-α inhibits NF-κB-dependent luciferase reporter plasmids. U-2 OS cells were treated as (A) but transfected with 1.5 μg of 3x κB and HIV-LTR luciferase reporter plasmids. (C) PFT-α inhibition of luciferase activity is dose dependent. U-2 OS cells were transfected with 1.5 μg of the indicated reporter plasmids and treated with the indicated concentrations of PFT-α for 24 hours. Cells were harvested and luciferase activity was measured and normalised for total protein. Results are expressed as fold repression above untreated transfected cells, and represent mean plus standard deviation of a minimum of 3 independent experiments. (D) PFT-α stabilises p53 protein and inhibits p53-dependent endogenous gene expression. p53 was induced in U-2 OS cells by co-transfection of 1.5 μg of ARF expression plasmid in the presence or absence of PFT-α, for 24 hours. Nuclear protein levels were prepared and analysed by western blot using the indicated anti-p53 and anti-p21 antibodies. (E) PFT-α inhibits NF-κB-dependent IκB-α reporter gene activity. HEK293 cells were co-transfected with 1.5 μg of IκB-luciferase and 1.5 μg of RSV-p65 or empty vector. Cells were then treated with 20 μM PFT-α for 24 hours after which cells were harvested and luciferase activity was measured. (F) PFT-α does not inhibit induction of endogenous IκB-α. Western blot analysis of endogenous IκB-α using whole cell lysates from cells subjected to equivalent conditions as the luciferase assay in (E).

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