Fig 3. Effects of XPO1 knock-down on IAV infection, cellular responses, and antiviral activity of the compounds.

A549 cells were transfected for 24 h with specific siRNA targeting XPO1 mRNA or nonspecific siRNA. Cells were then pretreated with the compounds (SEL, 1 μM; 4OI, 100 μM; BARD, 0.1 μM; SFN, 10 μM) for 12 h, infected with IAV PR8M (MOI = 1) for 2 h, and then incubated in fresh buffer containing the compounds for 22 h. A-C. Efficiency of XPO1 knock-down. A. XPO1 mRNA (RT-qPCR). B. XPO1 protein (immunoblot). C. Densitometry of B. D. Viral HA mRNA expression with reference to HPRT1 mRNA as internal control (RT-qPCR). E. Viral NP (immunoblot). F. IAV titers in cell culture supernatants (foci-forming assay, foci-forming units [FFU]/ml). G, H. IFIT1 and CXCL10 mRNA (RT-qPCR). I. Mitochondrial ROS (flow cytometry). J. Expression of NFE2L2, HMOX1, SLC7A11, AKR1B10, GCLM, and KEAP1 mRNAs (RT-qPCR, internal control HPRT1 mRNA). The heat map is based on log₂ fold change (scale as indicated in the color legend) with respect to expression in wild-type uninfected cells. Bar graphs for each target gene are shown in S2 Fig for additional clarity. n = 3, means ±SEM. One-way ANOVA with Tukey’s post-hoc test, using infected untreated wild-type or knock-down cells as reference. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.