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1.
Figure 7

Figure 7. IPS-1 is dispensable for induction of an antiviral state in response to long dsRNA molecules. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

IPS-1-/- and IPS-1 +/+ littermate controls were treated with the indicated amounts of dsRNA for 24h. Cells were then challenged with VSVgfp and GFP fluorescence intensity was measured at 24h pi.

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.
2.
Figure 4

Figure 4. Induction of Cxcl10, Ifit1, Irf7 and IFNb1 are more dependent on IRF3 following stimulation with short dsRNA molecules. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

WT and IRF3-/- MEFs were treated with ν200 (A) or v3000 (B) for 6 hours. Quantitative RT-PCR was performed in triplicate with gene expression being normalized to the housekeeping gene (GAPDH) and expressed as fold change relative to mock treated cells. Results were analyzed using a one-way ANOVA with a tukey post test, ** p<0.01, *** p<0.001.

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.
3.
Figure 1

Figure 1. WNv infection leads to the generation of intracellular dsRNA. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

A, Immunofluorescence microscopy of wild type (WT) MEFs infected with WNv strain Kunjin (MOI=10) or treated with poly IC (100 μg/mL) using the J2 anti-dsRNA antibody. B, Immunoblot analysis of Vero cells and WT MEFs infected with Kunjin virus (MOI 5 and 10, respectively) using the J2 anti-dsRNA antibody. The arrow indicates the running distance of a 3000 bp sized in vitro transcribed dsRNA molecule.

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.
4.
Figure 6

Figure 6. Evidence of a dsRNA length dependent antiviral response in the absence of IRF3 and type I IFN. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

IRF3&9-/- MEFs were treated with 8.5 nM of the indicated dsRNA for 24h. Cells were then challenged with VSVgfp, and GFP fluorescence intensity was measured at 24h pi. DsRNA length dependence was observed with ν3000 and poly IC but not with ν500 and ν1000 when a one way ANOVA was performed using a Dunnett's post test with ν200 being the control comparison. Results with poly IC in WT MEFs are shown for comparison purposes. * p<0.05, ** p<0.01.

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.
5.
Figure 5

Figure 5. The dependence on dsRNA length for antiviral state induction is exacerbated in the absence of IRF3. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

A, WT and B, IRF3-/- MEFs were treated with serially diluted nM concentrations of in vitro transcribed dsRNA of specific lengths (ν200, ν500, ν1000 and ν3000 bp) or poly IC for 6h. Cells were then challenged with VSVgfp and GFP fluorescence intensity was measured at 24h pi. EC50 values were determined as the dsRNA concentration providing 50% protection against VSVgfp. Length dependent antiviral responses were determined by performing a one- way ANOVA and a Dunnett's post test with ν200 being the control comparison. C, IRF-3 dependence was demonstrated by performing an unpaired T test, comparing WT to IRF3-/- EC50 values for each dsRNA length individually. * p<0.05, ** p<0.01.

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.
6.
Figure 3

Figure 3. ISG, IRF and IFN genes are induced in a dsRNA length-dependent fashion. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

A, WT, B, IRF3-/- and C, IRF3&9-/- MEFs were treated with in vitro transcribed dsRNA of different lengths (ν200, ν1000, ν3000) or poly IC using concentrations shown to induce a maximal antiviral response (1.5nM, 3nM and 8.5 nM dsRNA for WT, IRF3-/- and IRF3&9-/- MEFs, respectively) for 6h (WT and IRF3-/- MEFs) or for 24h (IRF3&9-/- MEFs). The fold change in transcript expression levels compared to levels in mock treated cells were measured using real time PCR arrays. These results are representative of two independent experiments. Transcript levels for the PCR array housekeeping genes (D) and IRF family members (E) were compared between mock treated WT, IRF3-/- and IRF3&9-/- MEFs. Differences in Ct values were not statistically significant, with the exception of β actin in IRF3&9-/- MEFs, which was found to be expressed at lower levels when compared to WT, but not IRF3-/-, MEFs. For each gene a one-way ANOVA was performed with a tukey post test, * p<0.05.

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.
7.
Figure 2

Figure 2. In vitro transcribed dsRNA of specific lengths derived from cloned fragments of the WNv genome is relatively stable within transfected cells. From: Long dsRNA induces an antiviral response independent of IRF3, IPS-1 and IFN.

A, DsRNA of 200 (lane 1), 500 (lane 2), 1000 (lane 3) and 3000 (lane 4) bp lengths was generated using the WNv genome as a template. Approximately 500 ng of each dsRNA was separated on a 1% agarose gel and visualized by ethidium bromide staining. As dsRNA runs slower than DNA, all dsRNA appear to be of the appropriate length when compared to a 1kb DNA marker (lane M). 4 μg of poly IC was also run on the gel (lane 5), with the average length determined to be approximately 4000 bp. B, WT MEFs were mock treated (M) or treated with 1 μg/mL poly IC (pIC) or in vitro transcribed dsRNA of four lengths (ν200, ν500, ν1000 or ν3000) for 6h, after which RNA was extracted. A dsRNA immunoblot was performed using 20μg RNA and dsRNA was detected using the J2 anti-dsRNA antibody. A ladder of 25 ng in vitro transcribed dsRNA was included to determine size (L).

Stephanie J. DeWitte-Orr, et al. J Immunol. ;183(10):6545-6553.

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