Journal: PLoS ONE

Article Title: Type-I Interferon is Critical for FasL Expression on Lung Cells to Determine the Severity of Influenza

doi: 10.1371/journal.pone.0055321

Figure Lengend Snippet: B6 mice were intranasally infected with 10 5 (closed triangle) or 10 2 (open square) pfu/head of the PR/8 virus. At 0, 3 or 5 DPI, the BALF of these mice were isolated. The amount of IFN- ß or total protein contained in these samples was assessed by mouse IFN- ß specific ELISA or BCA protein assay, respectively. The amounts of IFN- ß were normalized by that of the total protein in each sample. “N.D.” means not detected.

Article Snippet: The amount of IFN-β was assessed by mouse IFN-beta ELISA kit (R&D systems, Abingdon, UK).

Techniques: Infection, Virus, Isolation, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223 Promotes Type I Interferon Production in Antiviral Innate Immunity by Targeting Forkhead Box Protein O3 (FOXO3) *

doi: 10.1074/jbc.M115.700252

Figure Lengend Snippet: VSV-triggered type I IFN induces miR-223 expression in macrophages. A, RAW264.7 cells were infected with or without VSV at m.o.i. 0.1 for 12 h. Mouse peritoneal macrophages were infected with or without VSV at m.o.i. 10 for 24 h. The expressions of different miRNAs were measured by qPCR and normalized to the expression of U6 in each sample. B, RAW264.7 cells were infected with or without VSV at m.o.i. 0.1 for indicated times or at indicated m.o.i. for 12 h, and the expression of miR-223 was measured by qPCR and normalized to the expression of U6 in each sample. C, mouse peritoneal macrophages were infected with or without VSV at m.o.i. 10 for indicated times or at indicated m.o.i. for 24 h, and the expression of miR-223 was measured. D, RAW264.7 cells were infected with VSV at m.o.i. 0.1, and mouse peritoneal macrophages were infected VSV at m.o.i. 10. Total protein levels of VSV-G in lysates were detected by immunoblot at the indicated time. E, RAW264.7 were treated with suramin (200 μm) after infection with VSV at m.o.i. 0.1 for 1 h and then infected with VSV for 12 h; the expression of VSV RNA replicates, IFN-β and miR-223, were measured by qPCR, and total protein levels of VSV-G in lysates were detected by immunoblot. F, mouse peritoneal macrophages were treated with suramin (200 μm) after infection with VSV at m.o.i. 10 for 1 h and then infected with VSV for 24 h; the expression of VSV RNA replicates, IFN-β and miR-223, were measured by qPCR, and total protein levels of VSV-G in lysates were detected by immunoblot. G, RAW264.7 cells were infected with infectious VSV, heat- or UV-inactivated VSV at m.o.i. 0.1 for 12 h; IFN-β and miR-223 were measured by qPCR. H, mouse peritoneal macrophages were infected with infectious VSV, heat- or UV-inactivated VSV at m.o.i. 10 for 24 h; IFN-β and miR-223 were measured by qPCR. I, mouse peritoneal macrophages were infected with infectious H1N1, heat- or UV-inactivated H1N1 at m.o.i. 10 for 24 h; IFN-β and miR-223 were measured by qPCR. J, RAW264.7 macrophages were treated with rmIFN-β (25 ng/ml) for indicated times or pretreated with anti-mouse IFNAR1 antibody (10 μg/ml) for 2 h, then infected with VSV at m.o.i. 0.1 for indicated times, and the expression of miR-223 was measured. K, mouse peritoneal macrophages were treated as in E, infected with VSV at m.o.i. 10 for indicated times, and miR-223 expression was measured. Data are the mean ± S.D. (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. ***, p < 0.1; **, p < 0.01; *, p < 0.05; ns, not significant; Ctrl, control.

Article Snippet: Recombinant mouse IFN-β (rmIFN-β) was from Sino Biological (Beijing, China).

Techniques: Expressing, Infection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223 Promotes Type I Interferon Production in Antiviral Innate Immunity by Targeting Forkhead Box Protein O3 (FOXO3) *

doi: 10.1074/jbc.M115.700252

Figure Lengend Snippet: miR-223 positively regulates VSV-triggered type I IFN production. A, 0.5 ml of 2 × 105 RAW264.7 cells were transfected with control (Ctrl) mimics or miR-223 mimics (left), control inhibitors or miR-223 inhibitors (right) as indicated at a final concentration of 20 nm. After 48 h, miR-223 expression was measured. B, 0.5 ml of 2 × 105 mouse peritoneal macrophages were transfected as described in A, and after 48 h miR-223 expression was measured. C, 0.5 ml of 2 × 105 RAW264.7 cells were transfected as described in A. After 48 h, cells were infected by VSV at m.o.i. 0.1 for indicated times. IFN-α4 (left) and IFN-β (right) mRNA expression were measured by qPCR and normalized to the expression of β-actin in each sample. D, 0.5 ml of 2 × 105 mouse peritoneal macrophages were transfected as described in A. After 48 h, cells were infected by VSV at m.o.i. 10 for indicated times. IFN-α4 and IFN-β mRNA expression were measured by qPCR. IFN-β in supernatants was measured by ELISA. E, 0.5 ml of 2 × 105 peritoneal macrophages were transfected with control mimics or miR-223. After 48 h, cells were infected by VSV at m.o.i. 10 for indicated times. TNF-α and IL-6 mRNA expressions were measured by qPCR. TNF-α and IL-6 in supernatants were measured by ELISA. Data are the mean ± S.D. (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. ***, p < 0.1; **, p < 0.01; *, p < 0.05; ns, not significant.

Article Snippet: Recombinant mouse IFN-β (rmIFN-β) was from Sino Biological (Beijing, China).

Techniques: Transfection, Concentration Assay, Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-223 Promotes Type I Interferon Production in Antiviral Innate Immunity by Targeting Forkhead Box Protein O3 (FOXO3) *

doi: 10.1074/jbc.M115.700252

Figure Lengend Snippet: Antiviral function of miR-223 is mainly through targeting FOXO3. A and B, murine peritoneal macrophages were transfected with control siRNA and FOXO3 siRNA as indicated. After 48 h, FOXO3, IRF7, IFN-α4, and IFN-β mRNA expressions and FOXO3 protein levels were detected. C, murine peritoneal macrophages were transfected with control (Ctrl), miR-223 mimics, and FOXO3 siRNA, respectively, as indicated. After 48 h, macrophages were infected by VSV at m.o.i. 10 for 1 h and washed; intracellular VSV RNA replicates at indicated time points were quantified using qRT-PCR, and the value at 4-h time point in control cells served as 1-fold control. The relative IFN-β mRNA expression at indicated time points was detected by qRT-PCR, and IFN-β in supernatants was measured by ELISA. D, murine peritoneal macrophages were transfected with control inhibitor, miR-223 inhibitor, miR-223 inhibitors combined with control siRNA, and miR-223 inhibitors combined with FOXO3 siRNA, respectively, as indicated. After 48 h, macrophages were infected by VSV at m.o.i. 10 for 1 h and washed; intracellular VSV RNA replicates at indicated time points were quantified using qRT-PCR, and the value at 4 h in cells transfected with control inhibitors served as 1-fold control. E, RAW264.7 cells were transfected with FOXO3 overexpression plasmid. After 48 h, FOXO3 mRNA and protein levels were detected by qRT-PCR (left) and immunoblot (right). F, RAW264.7cells were transfected with control (Ctrl) mimics combined with control plasmid, miR-223 mimics combined with control plasmid, or control mimics combined with FOXO3 overexpression plasmid, and miR-223 mimics combined with FOXO3 overexpression plasmid. After 48 h, macrophages were infected by VSV at m.o.i. 10 for 1 h and washed; intracellular VSV RNA replicates at indicated time points were quantified using qRT-PCR, and the level at 4-h time point in cells transfected with control plasmid and control mimics served as 1-fold control. G, proposed positive regulatory loop of type I IFN/miR-223/FOXO3/IFR7 pathway in regulating antiviral innate immunity. Data are the mean ± S.D. (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. ***, p < 0.1; **, p < 0.01; *, p < 0.05; ns, not significant.

Article Snippet: Recombinant mouse IFN-β (rmIFN-β) was from Sino Biological (Beijing, China).

Techniques: Transfection, Infection, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Western Blot

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

Article Title: Loss of Mitochondrial Protease CLPP Activates Type I IFN Responses through the Mitochondrial DNA–cGAS–STING Signaling Axis

doi: 10.4049/jimmunol.2001016

Figure Lengend Snippet: (A–D) WT and CLPP-KO MEFs were cultured in the presence of regular media (Control) or media containing ddC (150μM). Cells were harvested after 4 days for (A) confocal microscopy with anti-DNA (DNA), anti-Tfam (mtDNA nucleoid marker) and anti-HSP60 (mitochondrial matrix protein). (B) Nucleoid quantification represented as percentage (%) of enlarged nucleoids (>450nm2). (C) Quantitative real-time PCR analysis of ISG expression (n=3 biological replicates). (D) Representative western blots of ISG protein expression. (E) Quantitative real-time PCR of ISGs in CLPP-KO MEFs transfected with control or siPolg2 siRNA for 72hrs. (n=3 biological replicates). (F) Quantitative real-time PCR of ISGs of WT and CLPP-KO MEFs treated with VBIT-4 (10μM) or Control (DMSO) for 48hrs (n=3 biological replicates). In (E), real-time PCR data are presented as relative expression percentage (%) setting siControl as 100%. Error bars represent mean ± s.e.m. of triplicates, Student’s t-test. In (C) and (F), error bars represent mean ± s.e.m. of triplicates, two-way ANOVA Tukey’s post-hoc. *p<0.05, **p<0.01, ****p<0.0001. All experiments are representative of 3–4 independent experiments.

Article Snippet: For VBIT-4 treatment plus challenge, cells were treated with VBIT-4 and 6hrs before the 48hrs harvesting time. mIFNβ (8234-MB-010/CF, R&D) was added to the cells at a final concentration of 1ng/ml.

Techniques: Cell Culture, Confocal Microscopy, Marker, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection