Figure 1.
Time course of the down-regulation of MAVS by polyI:C.
A549 (blue), HeLa (red) and 293 (green) cellswere transfected with polyI:C (500 ng/well). Quantitative RT-PCR for IFN-β and MAVS was performed. Data are presented as the mean ± SD of three independent experiments.
Figure 2.
Both ds- and ss-RNA induce MAVS mRNA degradaion.
Four hours after the transfection, the mRNA levels of IFN-β (A) and MAVS (B) were determined using quantitative RT-PCR. Data are presented as the mean ± SD of three independent experiments. *, p<0.01.
Figure 3.
Influence of MAVS mRNA on viral infection.
(A, C) HeLa cells were infected with mock-treated or SeV for 9 h. (B) HeLa cells were infected with mock-treated, IAV, or NDV. Quantitative RT-PCR for (A, B) MAVS and (C,D) IFN-β was performed. Data are presented as the mean ± SD of three independent experiments. *, p<0.01.
Figure 4.
MAVS mRNA decays rapidly in response to polyI:C transfection.
A549 cells were pretreated with EU 24 h before polyI:C transfection. At the indicated time points after transfection, EU-labeled mRNA was isolated and quantified using quantitative RT-PCR. Data are presented as the mean of three independent experiments.
Figure 5.
Involvement of TLR3, RLRs and IRF3 in the polyI:C-induced degradation of MAVS mRNA.
A549 cells were transfected with siRNA against TLR3, RIG-I, MDA-5 or IRF3 or with a control (scrambled) siRNA. Forty-eight hours after transfection, polyI:C was introduced into the cells for 4 h. The MAVS mRNA level was analyzed using quantitative RT-PCR. Data are presented as the mean ± SD of three independent experiments.
Figure 6.
Protective effect of IRF3 on the MAVS protein.
(A) A549 cells were transfected with polyI:C and incubated for up to 8 h. (B) Following IRF3 knockdown, A549 cells were transfected with polyI:C. Cell extracts were subjected to either SDS- (A and B) or native PAGE (A) and then blotted with anti-IRF3, anti-MAVS or anti-actin antibodies, as indicated. The results are representative of three independent experiments.
Figure 7.
Protein stability of MAVS in response to polyI:C transfection.
A549 cells were transfected with polyI:C for 1 h, followed by treatment with cycloheximide (CHX) for up to 24 h (illustrated as (A)). Cell extracts were analyzed by immunoblotting. The intensities of the immunoblot bands were quantified by Image J. Data are presented as the means of three independent experiments (B) or are representative of three independent experiments (C). The arrow indicates the cleaved MAVS protein.
Figure 8.
Effect of MAVS overexpression on polyI:C-induced IFN-β expression.
A MAVS-expressing plasmid or an empty plasmid (mock) was transfected into A549 cells for 24 h before polyI:C transfection. The mRNA levels of MAVS (A), GAPDH (B) and IFN-β (C) were determined using quantitative RT-PCR. Data are presented as the mean ± SD of three independent experiments. *, p<0.01.