Figure 7.

Kinetic profile of TNFα-stimulated luminescence in clonal HiBiT-edited E-selectin TERT2-HUVECs

To monitor the real-time kinetics of TNFα-induced expression of E-selectin, experiments were undertaken with the homozygous TERT2-HUVEC HiBiT E-selectin clone C8. As these kinetic experiments were performed over 15 h, the long acting caged nanoluciferase substrate endurazine was used.

(A) Time course of HiBiT E-selectin expression over 15 h in the continued presence of 50 nM LgBiT and endurazine. Values represent mean ± S.E.M from five independent experiments.

(B) Incubation with 1 nM TNFα in the continued presence of 50 nM LgBiT and endurazine yielded a significant increase in luminescence over 8 h (∗∗∗p < 0.001; paired t test of 5 independent experiments).

(C) To investigate potential depletion of LgBiT through the experiment, experiments were either performed in the continuous presence of 50 nM LgBiT incubation or following a 20 min addition of LgBiT (50 nM) at the end of the 15 h incubation with 1 nM TNFα. There was no significant difference between the luminescence values obtained.

(D) Time course of HiBiT E-selectin expression in response to increasing concentrations of TNFα. Values represent mean ± S.E.M from six (or five, 0.25 nM) independent experiments.

(E) Peak responses from (D) expressed as a percentage of the response to 1 nM TNFα.

(F) Log peak responses to increasing concentrations of TNFα. Data taken from (E). ∗∗∗∗p < 0.0001 (one-way ANOVA with Dunnett multiple comparison test). Symbols show the individual means in 5 or 6 independent experiments.