Fig 1: Depletion of NLRC5 or NLRX1 enhances the type I IFN production of GEN2.2 cells but does not influence the NF-?B pathway activity in response to VSV infection. (A–E) Cells were transfected with siRNAs specific for NLRC5, NLRX1 or scrambled (scr) siRNAs for 24 h. (A,B) After silencing cells were pre-treated with 0.25 µM CpG-A (pre-CpG-A) for 16 h to induce the cytosolic expression of RLRs. Following thorough washing steps cells were infected with VSV at the indicated MOIs. The protein levels of IFN-a, IFN-ß (A), TNF, IL-6, and IL-8 (B) were measured by ELISA after 18 h. (C–E) After silencing GEN2.2 cells were exposed to VSV at the indicated MOIs without CpG-A pre-treatment and the protein levels of RIG-I and MDA5 were detected by western blot at 24 h (C). Concentrations of IFN-a, IFN-ß (D), TNF, IL-6, and IL-8 (E) were measured by ELISA from the supernatant of the VSV-infected cells. (C) A representative blot is shown. Data are represented as means ± SD of 4 individual experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001 vs. untreated; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, n.d., not determined.
Fig 2: Impacts of CMPK2 knockdown or overexpression on DENV infection-induced effects(A-G) BMDMs, BMDCs, or human DCs were treated with small interfering RNA (siRNA) targeting CMPK2 to KD the expression of CMPK2 or control siRNA. Cells were then infected with DENV (MOI = 0.1 for murine cells and MOI = 5 for human DCs) for 48 h (murine cells) or 24 h (human DCs), and viral RNA and CMPK2 mRNA levels were measured (A for BMDMs, B for BMDCs, and C for human DCs). A549 cells were transfected with CMPK2-GFP or a GFP control and then infected with DENV, and the expression of CMPK2-GFP and viral NS2B was measured by western blotting (D). A549 cells were transfected with different doses of CMPK2-GFP or the GFP control and then infected with DENV (MOI = 1) or left uninfected, and the expression of CMPK2-GFP and viral NS2B was measured by western blotting (E). A549 cells with CMPK2 KD by treatment with siRNA were infected with DENV (MOI = 1) in the presence or absence of different dosages of IFN-a as indicated. The expression of CMPK2 and viral NS2B was measured by western blotting (F). BMDCs infected with DENV for 48 h were collected, and cell survival was measured with a CCK-8 assay (G). Values represent the mean of the individual measurements in each sample ±SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. p value was calculated by the Student's t test (A, B, C, D, and G) or one-way ANOVA (E).
Fig 3: FEZ1 regulates ISG expression in microglia cells(A and B) siRNA-mediated depletion of FEZ1 increases expression of ISGs (MxA, MxB, PKR, and ISG56) in CHME3s infected with HSV-1 (A) or VacV (B) as detected by the expression of viral proteins (HSV1: ICP4, ICP, ICP5; VacV: I3, G8, A25). L.E., long exposure; S.E., short exposure.(C) KO of FEZ1 (FEZ1 1–4 or FEZ1 pooled), but not non-targeting (NT) gRNAs, increases ISG levels in CHME3s.(D) Quantification of the ISG levels relative to HSPA8 in FEZ1 KO CHME3s from (C). Data are presented as the ratio to the difference between control and treatment groups.(E) WB analysis showing effects of FEZ1-Flag or S58A-Flag on expression of ISGs in CHME3s.(F) Quantification of ISG levels relative to HSPA8 in control, FEZ1-Flag, or FEZ1-S58A-Flag expressing CHME3s from (E). Statistical significance is presented as “A” when it was calculated for all groups using one-way ANOVA followed by Dunnett post-hoc test or as “t” if Student’s t test was applied to compare pairwisely Flag or FEZ1-S58A-Flag with FEZ1-Flag.(G and H) Measurement of IFN-ß levels in culture medium of CHME3 expressing Flag, FEZ1-Flag, and S58A-Flag by ELISA (G). Culture medium of CHME3s spiked with IFN-ß was included as positive control. Note that samples (except for the spiked control) were concentrated in order to obtain readings within the range of the standard curve (H) and are divided accordingly to present the actual values shown. One-way ANOVA followed by Tukey post-hoc test was used to calculate statistical significance in (D and G). (D, F and G) n = 3; red line, mean; bars, SD.See also Figure S1.
Fig 4: NLRX1 but not NLRC5 affects the specific RIG-I agonist-induced type I IFN and pro-inflammatory responses in human moDCs. (A–E) moDCs transfected with the indicated siRNAs were stimulated with the RIG-I ligand 5'ppp-dsRNA (RIGL, 1 µg/ml). The mRNA expression levels of IFNA1 and IFNB were assessed by real-time PCR after 12 h (A) and IFN-a, IFN-ß (B), TNF, IL-6, and IL-8 (C) protein levels were measured by ELISA after 24 h. (D,E) Kinetics of I?Ba degradation was determined by western blotting. (D) A representative blot is shown. (E) Bar graphs show the relative density of I?Ba measured at 60 min of stimulation. (A-C, E) Data are shown as mean ± SD from 4 independent experiments and analyzed using one-way ANOVA followed by Bonferroni's post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.01 ****p < 0.0001 vs. untreated; #p < 0.05, ###p < 0.001, ####p < 0.0001, n.d., not determined.
Fig 5: The effects of IFN-a receptor or STAT1 KO on DENV-induced CMPK2 expression and related events(A-E) BMDCs were prepared from mice with KO of IFN-aR or STAT1 or control mice. Cells were then infected with DENV (MOI = 1), and the expression of CMPK2, NS2B, phosphorylated STAT1, total STAT1, IRF1, and ß-actin was measured by western blotting (A). The expression of CMPK2 mRNA and DENV RNA was determined by qPCR (B). In addition, the production of mtROS (C) and 8-OHdG (D) was measured. Similar to Figure 4, the effects of IFN-aR or STAT1 KO on DENV-induced mtDNA release into the cytosol were determined in BMDCs (E). Values represent the mean of the individual measurements in each sample ±SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. p value was calculated by the Student's t test (B, C, D, and E).
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