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).

PFT-α inhibits active and recombinant firefly luciferase. (A) Exogenously added PFT-α inhibits cell extracts containing luciferase activity in vitro. HFF cells were transiently transfected with 1.5 μg of PG13 reporter plasmid. Cellular extracts were obtained, serial dilutions of PFT-α were added as indicated, and luciferase activity was measured. Results are expressed as the mean plus standard deviation of a minimum of 3 independent experiments. (B) PFT-α inhibits the activity of purified recombinant luciferase protein. Recombinant firefly luciferase was incubated with luciferase substrate in the presence or absence of 20 μM PFT-α and luciferase activity was measured. Results are expressed as light units, and represent mean plus standard deviation of a minimum of 3 independent experiments. Error bars are beneath the resolution of the graph and although calculated, cannot be seen. (C) PFT-α inhibits the activity of purified recombinant luciferase protein independently of substrate concentration. Recombinant firefly luciferase was incubated with increasing concentrations of luciferase substrate in the presence or absence of 20 μM PFT-α and luciferase activity was measured. Results are expressed as light units, and represent mean plus standard deviation of a minimum of 3 independent experiments. Luciferase substrate concentration is not provided by the manufacturer (Promega) and therefore is expressed as μl of substrate used.

PFT-α does not inhibit Renilla luciferase or CAT reporters in vivo. (A) PFT-α does not inhibit Renilla luciferase. The indicated cell lines were transfected with 1.5 μg of pRL-CMV-luciferase reporter and treated with 20μM 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 does not inhibit CAT reporter plasmids. U-2 OS cells were transfected with 5 μg of indicated reporters and treated with 20 μM PFT-α for 24 hours. Cells were harvested and CAT activity was measured and normalised for total protein. Results are expressed as percentage of conversion. Since there was a significant variation in the levels of activity of these reporters even in unstimulated conditions, although the overall conclusions were the same, the data is presented is a single experiment representative of 3 independent experiments.