Figure 3.

Expression of CD2v in PK15 cells induces IFN-β transcription in a NF-κB dependent manner. PK15 cells were transfected with pCD2v-HA or controls (pORFV120-Flag, pORFV113-Flag or Poly I:C), processed for immunofluorescence using primary antibodies against HA, Flag or NF-κB-p65 and secondary antibodies Alexa fluor 488 to detect CD2v, ORFV120 or ORFV113 and Alexa fluor 594 to detect NF-κB-p65 and counterstained with DAPI. Approximately 100 cells were counted/slide and results are shown as the mean values from three independent experiments. (A) Percentage of NF-κB-p65 nuclear translocation following the different treatments. p-values relative to controls ORFV120 and ORFV113 were 0.056 and 0.009 (3 h); 0.01 and 0.0006 (6 h); 0.019 and 0.02 (12 h); 0.005 and 0.004 (20 h); 0.016 and 0.009 (24 h), respectively. * and # denote statistical significance compared to ORFV120 and ORFV113, respectively. (B) Confocal microscopy images showing NF-κB-p65 nuclear translocation at 3 h pt (arrows). Green, CD2v or ORFV120 or ORFV113; Red, NF-κB-p65; Blue, DAPI. (C) Mean fluorescence intensity (MFI) of phosphorylated NF-κB (S536) measured by flow cytometry in CD2v and ORFV113 expressing cells at 3 h pt. Results are representative of three independent experiments. p-value relative to ORFV113 is 0.008. (D) PK15 cells pretreated with the NF-κB inhibitor parthenolide (1 μM) or DMSO (vehicle control) for one hour were transfected with pCD2v-HA or Poly I:C, fixed at 3 h pt and processed for immunofluorescence with antibodies against HA and NF-κB-p65. Approximately 100 cells were counted/slide and results are shown as mean values from three independent experiments (p = 0.001). (E) PK15 cells treated with parthenolide (1 μM) or DMSO for one hour were transfected with pCD2v-HA or pEmpty-HA in the presence or absence of parthenolide and IFN- β transcription assessed by RT-PCR at 6 h pt. Fold changes relative to Empty-HA and data are means from four independent experiments (p = 0.008). (*, p < 0.05; **, p < 0.01.)