Fig 1: EBV-encoded miRNAs regulate the expression of cellular genes and proteins involved in type I IFN activation. (A to E) 293T cells were cotransfected with the indicated wild-type luciferase reporter plasmids (WT) for RIG-I/DDX58 (A), IRAK2 (B), Viperin/RSAD2 (C), FYN (D), and OAS2 (E) or reporter plasmid in which the predicted seed sequence was mutated (mut) together with or without an miRNA-encoding plasmid. The luciferase expression in these cells was assessed and normalized to lysates from cells cotransfected with the wild-type 3′-UTR reporter and empty miRNA expression plasmid (Ø). P values were calculated using the one-way analysis of variance (ANOVA) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (F) Human primary B lymphocytes were infected with wild-type EBV (r_wt/B95.8) or EBV devoid of its miRNAs (r_ΔmiR). Five days postinfection, EBV-infected cells were counted and seeded at the same density. Four days later, the cells were lysed and the lysates were subjected to quantitative Western blot analysis using the Western blot stain-free TGX Bio-Rad normalization approach (Bio-Rad). Blots were probed with antibodies against IPO7, Viperin/RSAD2, TLR9, TLR7, FYN, RIG-I/DDX58, and OAS2. Protein levels were quantified and used to normalize the levels of the specific protein signals. Ratios of protein levels in cells infected with wild-type virus versus cells infected with mutant virus are shown. Reported are the results of four independent biological replicates.
Fig 2: Astrocytic TDP-43 alterations increase hippocampal IFN-inducible chemokines and other antiviral response factors.(A) Hippocampal RNA levels for indicated genes in 11-month-old littermate NTG controls, single transgenic hGFAP-tTA (tTA) and tetO–hTDP-43–?NLS (tetO) controls, and double transgenic hTDP-43–?NLS mice (?NLS). Transgenic tau-P301S mice (P301S) and their littermate controls at 10 months of age were used for validation and comparison of gene expression. (B and C) RNA levels from indicated brain regions as measured by RT-qPCR. Neocortex (CTX), hippocampus (HP), striatum (STR), and thalamus (THAL). Two-way ANOVA: F(9,52) = 2.06, P = 0.051 for interaction and F(3,52) = 3.061, P = 0.019 for genotype (B); F(9,49) = 1.65, P = 0.13 for interaction and F(3,49) = 0.85, P = 0.47 for genotype (C); Dunnett’s post hoc test: **P < 0.01 and ***P < 0.001 versus tetO. (D and E) CXCL10 immunoreactivity (green) in the dentate gyrus molecular layer and CA1 of 11-month-old NTG and ?NLS mice. Astrocyte markers GFAP or glutamine synthetase (GS), neuronal marker NeuN, or microglial/macrophage marker Iba1, as indicated. Yellow indicates overlay of green and red channels. DAPI (blue) was used to visualize nuclei. Insets in (D) show magnified views. Scale bars, 100 µm (D) and 20 µm (E). (F and G) Hippocampal RNA levels in 11-month-old NTG controls, single transgenic tetO and tTA controls, and ?NLS mice. One-way ANOVA: F(3,15) = 4.19, P = 0.024 (Ddx58); F(3,14) = 4.02, P = 0.029 (Ifih1); F(3,13) = 5.097, P = 0.015 (Eif2ak2); F(3,15) = 3.38, P = 0.046 (Rsad2); and F(3,15) = 3.29, P = 0.049 (Irf7). Dunnett’s post hoc test: *P < 0.05 versus tetO. (H) Viperin (green), CXCL10 (red), and glutamine synthetase (white) immunoreactivity in the dentate gyrus of 11-month-old NTG and ?NLS mice. Scale bar, 20 µm.
Fig 3: Effects of overexpression of viperin on Shaan virus replication(A) HEK293, A549 and MARC-145 cells were transfected with pHis-RSAD2, pMHis-RSAD2 or pHis-NCV for 24h and then cells were lysed to detect viperin by Western blot. Uncropped images of western blots displayed in Supplementary Fig. 1E. Transfected cells were infected with Shaan virus at MOI of 0.1 for the indicated times. Released (B) and cell-associated virus (C) were collected at the indicated time points and titrated in triplicate on MARC-145 cells. P values of <0.05 considered statistically significant (n = 3).
Fig 4: Therapeutic injection of STAT3 inhibitor reduces viral replication and AP and VMC pathology in mice. (A) Schematic map of HJC0152 treatment experiment. Mice were treated i.p. with 12.5 mg/Kg HJC0152 24 h before and 24 h after 103 pfu CVB3 infection. (B) Survival curve and weight loss curve in STAT3 inhibitor (HJC0152)-treated mice were followed by 7 dpi. (C) Immunoblotting analysis of day 3 pancreas or heart total lysates for VP1 expression. (D,E) Total RNAs from CVB3-infected day 3 pancreases were subjected to RT-QPCR using primers for IFN-α, IFN-β (D), ISGs (lfit1, Oas1, Mxa, Rsad2, Isg15 and Cxcl10) (E) and GAPDH. Relative mRNA was calculated by normalizing the values of the indicated genes to that of GAPDH. (F) Protein levels of MxA and Viperin from day 3 pancreases were quantitated by densitometric analysis and normalized to GAPDH, and they are presented as fold changes compared with the control. (G) Representative hematoxylin–eosin (H&E)-stained pancreas and heart sections from infected mice. Arrows indicate lymphocyte infiltration. Scale bar: 100 µm. (H) IL-1β, IL-6, TNF-α and IL-17A levels in day 7 pancreas homogenates were measured by ELISA. Data of (C–H) are represented as mean ± SEM (n = 5) of three independent experiments. ns, not significant. ***, p < 0.001; **, p < 0.01; *, p < 0.05.
Fig 5: Dampened M1 macrophage pro-inflammatory response is a hallmark of PolgD257A mutator mice during Mtb infection. Lung Il1ra (A), Isg15 (B), Rsad2 (C), Tnf (D), Nos2 (E), Ifng (F) Nlrp3 (G), Cxcl9 (H), and Cxcl10 (I) transcripts in WT and PolgD257A mice analyzed by qRT-PCR at day 21 post-Mtb infection. Transcripts were normalized to Actb transcript levels. (J) Immunoblot analysis of NLRP3, VIPERIN, and IL-1β protein levels in WT and PolgD257A mouse lungs at day 21 post-Mtb infection. GAPDH and ACTIN were used as loading controls. Statistical analysis: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance was determined for panels A–I using Mann-Whitney U test.
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