Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection
Fig 2
Defective optineurin causes autophagy failure.
(A) (upper panels) Fluorescence images of WT and Optn-KO MEFs stained with the autophagy marker, CYTO-ID. Scale bars, 50 μm. (lower panel) Fluorescence intensities of WT and Optn-KO MEFs stained with CYTO-ID were measured by flow cytometry and the results were compared. The black line indicates the unstained control. (B) Fluorescence intensities of WT (n = 4) and Optn-KO (n = 4) MEFs in steady and virus-infected states were measured by flow cytometry and the averages of the median fluorescence intensities are indicated. (C) LC3-I, LC3-II, optineurin, and actin of WT and Optn-KO MEFs in steady and virus-infected states were examined by western blotting. (D) IFNβ production by parental (n = 4) and two Optn-KO clones (n = 4 each) of GFP-LC3-RFP-LC3ΔG MEFs was measured by ELISA at 14 hours after inoculation. (E) Autophagy flux after infection is shown as a decreased GFP/RFP ratio in parental (n = 4) and two Optn-KO clones (n = 4 each) of GFP-LC3-RFP-LC3ΔG MEFs infected with SeV (Cantell strain). The GFP and RFP signal intensities in parental and two Optn-KO clones of GFP-LC3-RFP-LC3ΔG MEFs infected with SeV (Cantell strain) or mock treated were measured by flow cytometry. The GFP/RFP ratios were calculated using the median GFP and RFP signal intensities in each sample. The decreased ratios were calculated by comparing GFP/RFP ratios of virus-infected and mock samples. (F) Relative viral DI genome copy numbers in SeV-infected WT MEFs treated with wortmannin (n = 3) or DMSO (n = 3) at the indicated concentrations for 1.5 hours were measured by qPCR. Data are presented as mean values ± SD. Two-way ANOVA followed by the Tukey–Kramer method (B and F) and one-way ANOVA followed by Dunnett’s test (D and E) were applied for statistical analyses. **p < 0.01, ***p < 0.001.