Fig 2: Downregulation of NRF2-mediated antioxidant response improves influenza virus infection. (A) Western blot analysis of NRF2 and G6PD protein expression in A549 cells infected with different MOI (3, 0.3 and 0.03) of PR8 for 24 h. Actin was used as loading control. Data are the mean ± S.D. of two independent experiments, each condition run in duplicate (n = 4) (*P < 0.05 and **P < 0.001 vs. Mock). (B) ICW of viral proteins expression in A549 wild-type (IV) and A549 NRF2 -/- cells (IV NRF2 -/-) (left panel). The images show viral proteins expression (green fluorescence, anti-Flu) and cell monolayer (red fluorescence, CELL TAG). The graph represents the percentage (%) of RFU obtained from viral proteins normalized to CELL TAG. The analysis was performed measuring the RFU values of 4 different fields/well for each condition from the three experiments performed. Data are expressed as mean ± S.D. (*P < 0.05 vs. IV). Viral titer measured in the supernatants of A549 wild-type (IV) and A549 NRF2 -/- cells (IV NRF2 -/-) by Hemagglutination (HAU) and TCID50 assays (right panel). (C) Western blot analysis of G6PD expression in NRF2 -/- or wild type A549 cells infected with PR8 virus (IV conditions) for 24 h. Densitometry analysis of data obtained from two experiments performed; the values are expressed as mean ± S.D. (**P < 0.001, Mock vs. Mock -/-; *P < 0.05, IV vs. IV -/-). In the right panel is represented the analysis of NRF2 protein expression in the nuclear extracts of silenced G6PD or normal cells infected or not with PR8 virus (IV), left panel. Lamin was used as loading control. Densitometry analysis of data obtained from the two experiments performed; the values are expressed as mean ± S.D. (**P < 0.001, IV vs. Mock; *P < 0.05, IVsi vs. IV).

Fig 3: G6PD is associated with the MAVS/TRAF6 complex.a HEK293 cells were transfected with indicated plasmids for 48 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. b THP-1 cells were mock-infected or infected with VSV (MOI = 1) for the indicated times and subjected to Co-IP and immunoblotting analysis with the indicated antibodies. c HEK293 cells were transfected with indicated plasmids for 48 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. d THP-1 cells were transfected with vector control or Flag-G6PD for 36 h and infected with VSV (MOI = 1) for 6 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. e HEK293 cells were transfected with indicated plasmids for 48 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. f THP-1 cells were transfected with vector control, Flag-G6PD, si-ctrl, or si-TRAF6 for 36 h. Co-IP and immunoblot analyses were performed with the indicated antibodies. g THP-1 cells were transfected with vector si-ctrl, si-MAVS, or si-TRAF6 for 36 h and infected with VSV (MOI = 1) for 6 h, followed by an analysis of G6PD dimerization. All experiments were repeated at least three times. See also Supplementary Fig. 13. Source data are provided as a Source Data file.

Fig 4: G6PD protein reduction is mediated by a drop in the NRF2-mediated antioxidant response during the infection. (A) RT-qPCR analysis of NRF2 and G6PD gene expression during a time course (from 2 to 24 h p.i.) of PR8 virus infection. The analysis of NRF2 (blu line) and G6PD (red line) mRNA levels normalized to those of 18S (control) was performed in two independent experiments, each performed in duplicate (n = 4). Values are expressed as the mean ± S.D. (*P < 0.05 vs. 0 h, Mock-infected cells). (B) Western blot analysis of NRF2 (blu line) and G6PD (red line) protein synthesis during a time course (from 2 to 24 h p.i.) of PR8 infection. Actin was used as loading control. The densitometry analysis of values was obtained from two independent experiments, each sample run in duplicate. Data are the mean ± S.D. (n = 4) (*P < 0.05 and **P < 0.001 vs. Mock).

Fig 5: Evaluation of the PPP and SGS in human HCC. (a) Gene expression of G6PD, 6PGD, PHGDH, and PSAT1 was determined in HCC tumour tissue (n = 371) and normal liver tissue (n = 50). Boxplots show median represented by the horizontal line and the box boundaries represent the first and third quartile range. The whiskers are derived from the Tukey method. (b) Patients were sorted into percentiles of low, medium, and high expression of G6PD (low n = 38, medium n = 293, high n = 39), 6PGD (low n = 38, medium n = 292, high n = 40), PHGDH (low n = 38, medium n = 292, high n = 40), and PSAT1 (low n = 38, medium n = 191, high n = 40), and graphs represents overall patient survival in low, medium, and high expression groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 6PGD 6-phosphogluconate dehydrogenase, G6PD glucose-6-phosphate dehydrogenase, PHGDH phosphoglycerate dehydrogenase, PSAT1 phosphoserine aminotransferase 1.