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.
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