Fig. 2: USP18 positively regulates RLR-induced IFN-β signaling upon RNA virus infection. | Nature Communications

Fig. 2: USP18 positively regulates RLR-induced IFN-β signaling upon RNA virus infection.

From: USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS

Fig. 2

a, b qRT-PCR analysis of Ifnb (left), Ccl5 (middle), and ELISA analysis of IFN-β (right) from the culture supernatant of Usp18+/+ and Usp18−/− peritoneal macrophages infected with SeV (a) or EMCV (b) for indicated time points. (Representative data were collected and expressed as mean ± SD from three independent experiments. Two-tailed Student’s t test was performed, For a, left panel: ***p < 0.001, ***p < 0.001 in sequence, middle panel: *p = 0.0477, **p = 0.0029 in sequence, right panel: ***p < 0.001, ***p < 0.001 in sequence; For b, left panel: **p = 0.0032, middle panel: ***p < 0.001 in sequence, right panel: **p = 0.0013, ***p < 0.001 in sequence). c, d qRT-PCR analysis of Ifnb (left), Ccl5 (middle) mRNA, and ELISA analysis of IFN-β (right) from the culture supernatant of Usp18+/+ and Usp18−/− MEFs infected with SeV (c) or EMCV (d) for indicated time points. (Representative data were collected and expressed as mean ± SD from at least three independent experiments. Two-tailed Student’s t test was performed, For c, left panel: ***p < 0.001, **p = 0.0069, *p = 0.0068 in sequence, middle panel: **p = 0.0096, *p = 0.0145 in sequence, right panel: ***p < 0.001, ***p < 0.001 in sequence; For d, left panel: **p = 0.0028, *p = 0.0281 in sequence, middle panel: ***p < 0.001, ***p < 0.001 in sequence, right panel: ***p < 0.001. e qRT-PCR analysis of Ifnb from Usp18+/+ and Usp18-/- MEFs transfected with Poly I:C LMW (left) or Poly I:C HMW (right) for indicated time points. (Representative data were collected and expressed as mean ± SD from at least three independent experiments. Two-tailed Student’s t test was performed, left panel: **p = 0.0061, **p = 0.0014 in sequence, right panel: ***p < 0.001, ***p < 0.001 in sequence;) f Left panel, qRT-PCR analysis of Usp18 from Usp18+/+ cells pretreated with isotype antibody or α-IFNAR1 antibody followed by SeV infection for 8 h. Right panel, qRT-PCR analysis of Ifnb in Usp18+/+ and Usp18−/− cells pretreated with isotype antibody or α-IFNAR1 antibody followed by SeV infection for 8 h. (Representative data were collected and expressed as mean ± SD from at least three independent experiments. Two-tailed Student’s t test was performed, *p = 0.0154, *p = 0.0274 in sequence). g qRT-PCR analysis of Ifnb in Usp18+/+ and Usp18−/− MEF cells pretreated with or without mouse recombinant IFN-β followed by SeV infection for 8 h. (Representative data were collected and expressed as mean ± SD from at least three independent experiments. Two-tailed Student’s t test was performed, **p = 0.0025 *p = 0.0227 ***p < 0.001). h Immunoblot analysis of total and phosphorylated (p-) TBK1, total and phosphorylated (p-) IRF3 in lysates of Usp18+/+ and Usp18−/− MEFs infected with SeV for 0–12 h and harvested at indicated time points. For the densitometric analysis (right), bands were normalized with individual actin, and phosphorylated protein over the total amount of that protein was determined. i Native gel analysis (above) and SDS-PAGE (below) of IRF3 dimerization in lysates of Usp18+/+ and Usp18−/− MEFs infected with SeV for 0–8 h. Densitometry of dimer IRF3 versus IRF3 (right). Representative data were collected and expressed as mean ± SD from at least three independent experiments. Two-tailed Student’s t test was performed, with *p < 0.05, **p < 0.01, ***p < 0.001 for Usp18+/+ versus Usp18−/− (a–g).

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