Design and functional analysis of the NF-κB-TRE-Gluc reporter and TLR4-LOV construct. (A) Schematic representation of the engineered NF-κB-TRE-Gluc reporter, the TLR4, and the TLR4-LOV constructs. NF-κB-TRE-Gluc encompasses five tandem repeats (TRE), Gaussia Luciferase (Gluc), dTomato under the control of CMV promoter, and Hygromycin B (HYG B). TLR4-LOV consists of a full-length human TLR4 molecule with an incorporated LOV domain at the C-terminal intracellular domain. (B) Schematic representation of the function of TLR4-LOV. Homodimerisation and activation of NF-κB in response to blue light can be detected in real-time via measuring Gluc. (C) HeLa, (D) 293Ta, and (E) PANC-1 were transiently co-transfected with either TLR4 or TLR4-LOV with the NF-κB-TRE-Gluc reporter, and Gluc was measured 6 h post LPS (100 ng/mL) or light (470 nm, 7 V, 7.5 min) treatment, respectively, or left untreated (w/o). Post-ANOVA multiple comparisons relative to the control were performed using Dunnett’s test. Error bar = SD. n = 6; ** p < 0.01, *** p < 0.001.