Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

doi: 10.4049/jimmunol.1800377

Figure Lengend Snippet: FIGURE 1. Type I IFN inhibits SCRV replica- tion. (A) Miiuy croaker macrophages seeded in 24- well plates overnight were untreated or treated with 200 U/ml of IFN-a. After 12 h, macrophages were infected with equal amounts (MOI = 5) of SCRV for 48 h. Cells were observed for morphological changes and photographed by light microscopy at original magnification 3100. (B) Total SCRV RNA was extracted from the treated macrophages de- scribed above (A). The viral RNA levels were evaluated by qPCR. Data represent the averages of results from three independent experiments. The error bars indicate the SD of data from three in- dependent experiments. **p , 0.01.

Article Snippet: The expression analysis of miR-210 was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen Biotech) with the following conditions: 94 ̊C for 2 min, 40 cycles of two steps (94 ̊C for 20 s, 60 ̊C for 30 s, and 72 ̊C for 30 s).

Techniques: Infection, Light Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

doi: 10.4049/jimmunol.1800377

Figure Lengend Snippet: FIGURE 2. SCRV infection upregulates miR-210 expression in macrophages. Miiuy croaker macrophages were infected with SCRV (MOI = 5) at different times (A) or various SCRV MOIs for 18 h (B). The miR-210 expression levels were measured by qPCR. Data represent the averages of results from three independent experiments. The error bars indicate the SD of data from three independent experiments.

Article Snippet: The expression analysis of miR-210 was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen Biotech) with the following conditions: 94 ̊C for 2 min, 40 cycles of two steps (94 ̊C for 20 s, 60 ̊C for 30 s, and 72 ̊C for 30 s).

Techniques: Infection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

doi: 10.4049/jimmunol.1800377

Figure Lengend Snippet: FIGURE 4. miR-210 modulates SCRV-triggered type I IFN and inflammatory cytokines expression. (A) Miiuy croaker macrophages were transfected with negative control mimics (NC) or miR-210 mimics (miR-210) (left) and negative control inhibitors (NC-i) or miR-210 inhibitors (miR-210-i) (right) for 48 h, and then miR-210 expression was determined by qPCR. (B) Macrophages were infected with or without SCRV (MOI = 5) for 18 h. The mRNA levels of IFN-2, Mx1, ISG15, Viperin, TNF-a, IL-6, and IL-8 were determined by qPCR. (C–E) Macrophages were transfected with NC, miR-210, NC-i, or miR- 210-i. After 48 h posttransfection, the cells were infected with SCRV at MOI of 5 for 18 h. The expression level of IFN-2 (C), antiviral genes, including Mx1, ISG15, and Viperin (D), and inflammatory cytokines, including TNF-a, IL-6, and IL-8 (E), were analyzed by qPCR. Data represent the averages of results from three independent experiments. The error bars indicate the SD of data from three independent experiments. **p , 0.01, *p , 0.05.

Article Snippet: The expression analysis of miR-210 was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen Biotech) with the following conditions: 94 ̊C for 2 min, 40 cycles of two steps (94 ̊C for 20 s, 60 ̊C for 30 s, and 72 ̊C for 30 s).

Techniques: Expressing, Transfection, Negative Control, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

doi: 10.4049/jimmunol.1800377

Figure Lengend Snippet: FIGURE 6. miR-210 suppresses the expression of STING at both mRNA and protein levels. (A and B) Miiuy croaker macrophages were transfected with NC or miR-210 (A) and NC-i, or miR-210-i (B). After 48 h, the protein and mRNA levels of STING were determined by Western blot and qPCR, re- spectively. (C and D) HEK293 cells were cotransfected with STING expression plasmid, along with miR-210 (C) or pre–miR-210 plasmid (D) in a concentrate gradient manner. After 48 h, the protein and mRNA levels of STING were determined by Western blot and qPCR. NC (C) and pcDNA3.1 vector plasmid (D) were used to control the same amount of molecules for transfections. (E and F) After transfection of NC or miR-210 (E) and NC-i or miR-210-i (F) for 48 h, the macrophages were then treated with SCRV for 18 h. The mRNA expressions of STING were analyzed by qPCR. Data represent the averages of results from three independent experiments. The error bars indicate the SD of data from three independent experiments. **p , 0.01, *p , 0.05. miR-210-i, miR-210 inhibitor; NC, negative control; NC-i, negative control inhibitor.

Article Snippet: The expression analysis of miR-210 was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen Biotech) with the following conditions: 94 ̊C for 2 min, 40 cycles of two steps (94 ̊C for 20 s, 60 ̊C for 30 s, and 72 ̊C for 30 s).

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, Control, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

doi: 10.4049/jimmunol.1800377

Figure Lengend Snippet: FIGURE 8. Knockdown of STING attenuates the expression of IFN-2 and inflammatory cytokines. (A) Miiuy croaker macrophages were transfected with control siRNA (si-Ctrl) or si-STING. After 48 h, STING mRNA and protein levels were determined by qPCR and Western blot, respectively. (B–D) After 48 h transfected with si-Ctrl or si-STING, macrophages were then infected with SCRV for 18 h. The expression levels of IFN-2 (B), antiviral genes, including Mx1, ISG15, and Viperin (C), and inflammatory cytokines, including TNF-a, IL-6, and IL-8 (D) were determined by qPCR. Data represent the averages of results from three independent experiments. The error bars indicate the SD of data from three independent experiments. **p , 0.01.

Article Snippet: The expression analysis of miR-210 was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen Biotech) with the following conditions: 94 ̊C for 2 min, 40 cycles of two steps (94 ̊C for 20 s, 60 ̊C for 30 s, and 72 ̊C for 30 s).

Techniques: Knockdown, Expressing, Transfection, Control, Western Blot, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Rhabdovirus-Inducible MicroRNA-210 Modulates Antiviral Innate Immune Response via Targeting STING/MITA in Fish.

doi: 10.4049/jimmunol.1800377

Figure Lengend Snippet: FIGURE 9. miR-210 promotes SCRV replication. (A) Miiuy croaker macrophages were transfected with NC, miR-210, NC-i, or miR-210-i and infected with SCRV at MOI 5 for 1 h and washed and then added with fresh medium. After 72 h, SCRV TCID50 in cultural supernatants was measured with EPC cells. (B) The qPCR analysis was conducted for supernatant SCRV RNA. Data represent the averages of results from three independent experiments. The error bars indicate the SD of data from three independent experiments. **p , 0.01. (C) EPC cells seeded in 24-well plates overnight were treated with cultural supernatants at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. miR-210-i, miR-210 inhibitor; NC, negative control; NC-i, negative control inhibitor.

Article Snippet: The expression analysis of miR-210 was executed by using the miRcute miRNA qPCR Detection Kit (Tiangen Biotech) with the following conditions: 94 ̊C for 2 min, 40 cycles of two steps (94 ̊C for 20 s, 60 ̊C for 30 s, and 72 ̊C for 30 s).

Techniques: Transfection, Infection, Staining, Negative Control