Figure 5.

Selective Inhibition of Canonical JAK-STAT Signaling Might Lead to an Alternative Antiviral Signaling Network following CT-VT-RT Treatment

(A) Transcriptional profiles determined via qPCR reveal the downregulation of IFN-β and STAT1 upon inhibitor treatment, also affecting downstream ISGs (MX1 and ISG15) and DDX58. (B) Immunoblot analysis after CT-VT-RT regimen with or without signaling cascade inhibitors monitoring levels of STAT1 and phosphorylated STAT1, along with BATF2, IRF1, and β-tubulin as loading controls as indicated. (C) Loss of IFN-β production upon inhibitor treatment determined by ELISA. (D) Immunoblot analysis after CT-VT-RT regimen with inhibitors. There was no change in SAMD9 expression (upper panel), but abrogation of TRAIL expression (lower panel) is shown upon inhibition. β-tubulin served as the loading control. (E) Proximity ligation assay reveals potential molecular interaction of BATF2 with IRF1 observed through increased red amplification signal in cells treated with CT-VT-RT plus fludarabine. Basal signal was observed in CT-VT-RT and CT-VT-RT plus DMSO alongside IgGs (inset) in all treatment regimens. Scale bar, 20 μm. (F) Decreased but detectable CCL5 expression in samples treated with CT-VT-RT plus molecular inhibitors determined via ELISA. (G) Effector caspase 3 or 7 activity in samples treated with CT-VT-RT plus molecular inhibitors indicate alternative host machinery initiating apoptosis. One-way ANOVA with Dunnett’s multiple comparison test (A, C, F, and G); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Flud, fludarabine.