Figure 1.
Establishment of 293TLR3HA-derived IFN-β promoter and NFκB reporter cell lines.
To test the dose dependent response of 293TLR3/IFNβLuc (A) and 293TLR3/NFκBLuc (B) cells to TLR3 ligand, cells were mock treated or treated with the indicated concentrations of poly I:C for 16 h. Promoter activities were expressed as fold of induction of luciferase activity compared to mock treated control (mean +/− standard derivations, n = 3).
Figure 2.
Characterization of 293TLR3HA-derived IFN-β promoter and NFκB reporter cell lines.
293TLR3/IFNβLuc and 293TLR3/NFκBLuc cells were treated with 2 µg/ml of poly I:C in the absence or presence of the indicated concentrations of compounds for 16 h. (A) Endosome acidification inhibitor chloroquin. (B) NFκB pathway IKK-2 inhibitor. (C) PI3k inhibitor LY294002. Luciferase activities were determined and the effects of the tested compounds on the promoter activity were normalized and expressed as a percentage of luciferase activity obtained from the cells treated with poly I:C alone (mean +/− standard derivations, n = 3).
Figure 3.
RO 90–7501 selectively enhances the activation of IFN-β promoter but not NFκB reporter by poly I:C.
(A) Structure of RO 90–7501. (B) 293TLR3/IFNβLuc and 293TLR3/NFκBLuc cells were mock treated or treated with 10 µM of RO 90–7501, 2 µg/ml of poly I:C, alone or in combination for 16 h. Luciferase activities were normalized to mock-treated controls and expressed as fold of induction (mean +/− standard deviation, n = 8). P values were calculated using the two-tailed student t test. (C) 293TLR3/IFNβLuc and 293TLR3/NFκBLuc cells were mock treated or treated with 2 µg/ml of poly I:C in the absence or presence of the indicated concentrations of RO 90–7501 for 16 h. The effects of RO 90–7501 on the IFN-β promoter and NFκB reporter activation were normalized to mock-treated controls and expressed as fold of induction of luciferase activity (mean +/− standard derivation, n = 3). P values were calculated using the two-tailed student t test, with * indicates P<0.05 compared to poly I:C treated alone. (D) 293TLR3/IFNβLuc cells were mock treated or treated with indicated concentrations of RO 90–7501 for 16 h. Cell viability was determined by a MTT assay and the results were plotted as the dose-dependent absorbance values at 570 nm.
Figure 4.
RO 90–7501 does not alter the kinetics of IFN-β promoter and NFκB activation by poly I:C.
293TLR3/IFNβLuc and 293TLR3/NFκBLuc cells were treated with 2 µg/ml of poly I:C alone or in the presence of 10 µM of RO 90–7501. At the indicated time after treatment, the cells were lysed and luciferase activity was measured and expressed as fold of induction over untreated controls (mean +/− standard derivations, n = 3). P values were calculated using the two-tailed student t test, with * indicates P<0.05 compared to poly I:C treated alone.
Figure 5.
293TLR3/IFNβLuc cells were treated either with 2 µg/ml poly I:C alone or in combination with 10 µM of RO 90–7501 by the six different treatment schedules depicted on the left panel. Luciferase activities in the cell lysate were measured and expressed as fold of induction over poly I:C untreated controls (mean +/− standard derivations, n = 3). P values were calculated using the two-tailed student t test.
Figure 6.
RO 90–7501 enhances the expression of endogenous IFN-β gene in both 293TLR3HA and human macrophage cell line THP-1.
(A) 293TLR3HA cells were mock treated or treated with 10 µM of RO 90–7501, 2 µg/ml poly I:C, alone or in combination. The cells were harvested at the indicated time since start of the treatment. The levels of IFN-β and β-actin mRNAs were determined by semi-quantitative RT-PCR. (B) THP-1 cells were mock treated or treated with the indicated concentrations of poly I:C in the absence or presence of 10 µM RO 90–7501 for 6 h. The levels of secreted type I IFN were then determined with HEK-Blue IFN-α/β cells using culture media transferred from treated THP-1 cells. The level of ISG54 promoter driven SEAP expression in the supernatant was then determined with QUANTI-Blue and expressed as absorbance values at 655 nm, normalized to mock treated controls (mean +/− standard derivations, n = 8). P values were calculated using the two-tailed student t test. (C) 293TLR3HA cells were mock treated or treated with the indicated concentrations of poly I:C in the absence or presence of 10 µM RO 90–7501 for 6 h. Secreted IL-8 in culture media from treated 293TLR3HA cells were determined with a human IL-8 ELISA kit, and expressed as absorbance values at 450 nm (mean +/− standard derivations, n = 3).
Figure 7.
RO 90–7501 enhances the cytoplasmic RLR ligand-induced IFN-β expression.
(A) 293/IFNβLuc cells, which are deficient of TLR3, were transfected with poly I:C and mock treated or treated with the indicated concentrations of PI3K inhibitor LY294002 for 16 h. Luciferase activities in the cell lysate were measured and expressed as fold of induction over mock-transfected controls (mean +/− standard derivations, n = 24). (B) 293/IFNβLuc cells were either mock transfected (no RNA) or transfected with poly I:C. The cells were then mock treated or treated with 10 µM of RO 90–7501 for 16 h. Luciferase activities in the cell lysate were measured and expressed as fold of induction over mock-transfected controls (mean +/− standard derivations, n = 32). P values were calculated using the two-tailed student t test.
Figure 8.
RO 90–7501 enhances poly I:C-induced antiviral activity.
293TLR3HA cells were left untreated or treated with indicated concentrations of poly I:C in the absence or presence of 10 µM RO 90–7501 for 12 h. Cells were then infected with VSV at a MOI of 0.001. Culture media were harvested 16 h post infection and the virus yields were determined by a plaque assay and presented as number of plaque forming units (pfu) per milliliter medium (mean +/− standard derivations, n = 3). P values were calculated using the student t test.
Figure 9.
RO 90–7501 enhances poly I:C induced MAPK pathway activation.
(A) 293TLR3HA cells were mock treated or treated with 2 µg/ml poly I:C, 10 µM RO 90–7501, alone or in combination for 30 min. A fraction of the cell lysate was analyzed by Western blot for total, phosphorylated p38, ERK and JNK or β-actin on a LI-COR Odyssey system. (B) 293TLR3/IFNβLuc cells were treated with 2 µg/ml of poly I:C in the absence or presence of the indicated concentrations of MAPK pathway inhibitors for 16 h. Luciferase activities were determined and the effects of the tested compounds on the promoter activity were normalized and expressed as a percentage of luciferase activity obtained from the cells treated with poly I:C alone (mean +/− standard derivations, n = 3).