Figure 6.

Nrf2-Induced Autophagy Curtails Antiviral Innate Immunity

(A) 6 hr following transfection with increasing doses of an Nrf2 overexpression plasmid, HEK293T cells were infected with Sendai virus (SeV) (40 hemagglutinin [HA]/mL) for 18 hr and ISRE promoter activity was assessed by a luciferase reporter assay. (B) A549 cells were transfected with control or Nrf2 siRNA and 48 hr later were challenged with VSVΔ51-GFP (MOI 0.01) for an extra day. Whole-cell extracts were analyzed for Nrf2, VSV-GFP, LC3, P-STAT1, STAT1, RIG-I, and β-actin by immunoblotting. (C) 6 hr following transfection with increasing doses of an HO-1 overexpression plasmid, HEK293T cells were infected with SeV (40 HA/mL) for 18 hr and ISRE promoter activity was assessed by a luciferase reporter assay. (D) A549 cells were transfected with control or HO-1 siRNA and 48 hr later were challenged with VSVΔ51-GFP (MOI 0.01) for an extra day. Whole-cell extracts were analyzed for HO-1, VSV-GFP, LC3, P-STAT1, STAT1, RIG-I, and β-actin by immunoblotting. (E and F) A549 cells were transfected with control or Atg7 siRNA and 48 hr later were challenged with VSVΔ51-GFP (MOI 0.01) for an extra day. (E) VSVΔ51 infection was determined by flow cytometry. Data are the means ± SEM of three independent experiments. (F) Whole-cell extracts were analyzed for VSV proteins, RIG-I, P-STAT1, STAT1, and β-actin by immunoblotting. (G and H) U-2 OS cells were transfected with control or Nrf2 siRNA and 48 hr later were infected with VSVΔ51-GFP (MOI 0.001). (G) Viral infectivity was determined by flow cytometry 24 hr post-infection and observed using a fluorescent microscope. Data are the means ± SEM of three independent experiments. (H) Whole-cell extracts were also analyzed by immunoblotting for Nrf2, VSV proteins, LC3, P-STAT1, and β-actin.