Figure 3.
Increased AHR activity and TF and PAI-1 levels in endothelial cells treated with plasma from animals with xenografts. (A) Optimum concentration of mouse plasma for AHR activity. Endothelial cells stably expressing a xenobiotic responsive element promoter-luciferase reporter construct were treated for 24 hours with indicated concentrations of plasma from 3 athymic control mice (without xenograft). AHR activity was quantified by firefly luciferase units and normalized to amount of protein. An average of 3 independent experiments is shown. Error bars = SEM. Compared with cells treated with serum-free medium (control), P values were .04, .003, .02, and .03 for plasma concentration of 0.5%, 1%, 1.5%, and 2%, respectively. (B) Higher AHR-inducing activity of plasma from mice with a colon cancer xenograft. AHR activity assay was performed as noted earlier by using 1% plasma obtained from mice with a colon cancer xenograft. An average of 2 independent determinations performed in duplicate is shown. Plasma samples obtained from 8 mice per group as described in Figure 1A were used. Error bars = SEM. P = .007 for plasma from xenograft-bearing (HT-29) animals compared with matching controls (CTL). (C) Higher TF-inducing activity of plasma from mice with a colon cancer xenograft. Endothelial cells were treated for 24 hours with 1% plasma obtained from mice with a colon cancer xenograft with or without 20 µM of CH223191. Plasma from animals without tumors served as negative controls as described in Figure 1A. The cells were subjected to procoagulant TF activity assay by using Factor VIII and Factor XIa and chromogenic substrate (see Methods). An average of 2 independent experiments with plasma obtained from 8 animals per group performed in duplicates is shown. Error bars = SEM. *P = .019 compares CTL and HT-29 and P = .047 HT-29 and HT-29 + CH223191. (D) Kyn increases PAI-1 in an AHR-dependent manner. Endothelial cells treated with a titrated concentration of Kyn as shown with or without AHR inhibitor CH223191 for 24 hours. Using western blotting, equal amounts of lysates were probed separately on different blots for PAI-1 and β-actin (loading control) due to the proximity of the molecular weights. Representative immunoblots from 3 independent experiments are shown. Molecular weight ladder (kDa) is shown on the left of the blot. The PAI-1 band normalized to β-actin by using ImageJ is shown, and values obtained are noted below the PAI-1 blot. (E) Endothelial cells treated with 1% plasma from control and HT-29 mice as alluded in Figure 1A with or without 20 µM of CH223191 for 24 hours. The lysates from 3 separate representative mice are shown from a total 8 mice per group. Actin served as a loading control.

Increased AHR activity and TF and PAI-1 levels in endothelial cells treated with plasma from animals with xenografts. (A) Optimum concentration of mouse plasma for AHR activity. Endothelial cells stably expressing a xenobiotic responsive element promoter-luciferase reporter construct were treated for 24 hours with indicated concentrations of plasma from 3 athymic control mice (without xenograft). AHR activity was quantified by firefly luciferase units and normalized to amount of protein. An average of 3 independent experiments is shown. Error bars = SEM. Compared with cells treated with serum-free medium (control), P values were .04, .003, .02, and .03 for plasma concentration of 0.5%, 1%, 1.5%, and 2%, respectively. (B) Higher AHR-inducing activity of plasma from mice with a colon cancer xenograft. AHR activity assay was performed as noted earlier by using 1% plasma obtained from mice with a colon cancer xenograft. An average of 2 independent determinations performed in duplicate is shown. Plasma samples obtained from 8 mice per group as described in Figure 1A were used. Error bars = SEM. P = .007 for plasma from xenograft-bearing (HT-29) animals compared with matching controls (CTL). (C) Higher TF-inducing activity of plasma from mice with a colon cancer xenograft. Endothelial cells were treated for 24 hours with 1% plasma obtained from mice with a colon cancer xenograft with or without 20 µM of CH223191. Plasma from animals without tumors served as negative controls as described in Figure 1A. The cells were subjected to procoagulant TF activity assay by using Factor VIII and Factor XIa and chromogenic substrate (see Methods). An average of 2 independent experiments with plasma obtained from 8 animals per group performed in duplicates is shown. Error bars = SEM. *P = .019 compares CTL and HT-29 and P = .047 HT-29 and HT-29 + CH223191. (D) Kyn increases PAI-1 in an AHR-dependent manner. Endothelial cells treated with a titrated concentration of Kyn as shown with or without AHR inhibitor CH223191 for 24 hours. Using western blotting, equal amounts of lysates were probed separately on different blots for PAI-1 and β-actin (loading control) due to the proximity of the molecular weights. Representative immunoblots from 3 independent experiments are shown. Molecular weight ladder (kDa) is shown on the left of the blot. The PAI-1 band normalized to β-actin by using ImageJ is shown, and values obtained are noted below the PAI-1 blot. (E) Endothelial cells treated with 1% plasma from control and HT-29 mice as alluded in Figure 1A with or without 20 µM of CH223191 for 24 hours. The lysates from 3 separate representative mice are shown from a total 8 mice per group. Actin served as a loading control.

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