Journal: PLOS Pathogens
Article Title: Interferon-β induction heterogeneity during KSHV infection is correlated to levels and activation of the transcription factors ATF2 and RelA, and not IRF3
doi: 10.1371/journal.ppat.1013947
Figure Lengend Snippet: A-L. BC3-IFN-βp-tdTomato reporter cells were treated with 20 ng/ml TPA for 48 hours to induce the lytic cycle (“lytic (+TPA)”). Where indicated, the cells were also treated with 10 μM of the pan-caspase inhibitor IDN-6556 (“casp-i”), 10 μM of the TBK1 inhibitor MRT76307 (“TBK1i”), 10 μM of the cGAS inhibitor G140 (“cGASi”), and/or a cocktail of antibodies against type I IFNs and their receptor (“anti-IFN Abs”) at 1:2000 dilution. A-B. IFN-β and IFN-λ1 mRNA levels were measured by qRT-PCR and normalized to 18S rRNA. The expression levels are plotted relative to lytic+casp-i samples that did not receive TBK1i or cGASi treatment for each experiment. n = 3. **** = p < 0.0001. Two-way ANOVA followed by Tukey’s post hoc multiple comparisons test. C-L. Protein lysates were probed for phosphorylated IRF3 (Ser386 or Ser396), total IRF3, phosphorylated TBK1 (Ser172), total TBK1, and β-actin as a loading control. As a positive control for IRF3 and TBK1 activation/phosphorylation, A549 cells were treated with 7 μg/mL poly(I:C) for 6 and 48 hours before protein lysates were collected. Protein was isolated from treated BC3-IFN-βp-tdTomato reporter cells without sorting (“bulk”), or after sorting the lytic+casp-i ( C-G ) or lytic+casp-i+anti-IFN Abs ( H-L ) sample based on tdTomato expression. Western blot for phosphorylated TBK1 and phosphorylated IRF3 for bulk lytic+casp-i or lytic+casp-i+anti-IFN Abs treated samples and sorted tdTomato+ and tdTomato- samples were quantified by normalizing the density of phosphorylated protein to unphosphorylated protein bands. Phosphorylation levels are plotted relative to the bulk lytic+casp-i or lytic+casp-i+anti-IFN Abs treated samples. M. KSHV-negative BJAB cells were treated with vehicle, 20 ng/ml TPA, and/or 10 μM of the pan-caspase inhibitor IDN-6556 (“casp-i”) for 48 hours. Protein was isolated and stained for phosphorylated TBK1, total TBK1, phosphorylated IRF3 (Ser386), and total IRF3. Images shown are representative of 3 replicates. None of the differences in quantified levels are statistically significant based on one-way ANOVA followed by Tukey’s post hoc multiple comparisons test (D-E, G, I-J, L).
Article Snippet: Where indicated, A549 cells were seeded at 0.5x10 cells per well in a 6-well plate 1 day before treatment, and treated with vehicle (water) or 7 μg/mL poly(I:C) (poly(I:C) LMW/ LyoVec, Invivogen, TLRLPICWLV) for 6 or 48 hours.
Techniques: Quantitative RT-PCR, Expressing, Control, Positive Control, Activation Assay, Phospho-proteomics, Isolation, Western Blot, Staining
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