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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Gene expression analysis of Tlr7 (A), Irf-7 (B), Ifn-α (C), Isg-56 (D) to β-actin in whole brains from WNV-infected WT and Tlr8−/− mice at day 4 p.i. by qPCR (n = 6-8 per group). (E) Immunoblotting analysis of TLR7 (140 kDa), total STAT-1 (94/87 kDa) ISG-56 (56 kDa), total IRF-7 (51 kDa), and β-Tubulin (55 kDa) from whole brain lysates of WNV-infected WT and Tlr8−/− mice at day 4 p.i. (n = 6-8 per group). Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6–12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr. Gene expression of Tlr7 (F), Irf-7 (G), Ifn-α (H), Isg-56 (I), and WNV-E (J) to β-actin were measured by qPCR. (K) WNV-infected neurons probed with anti-WNV-E (green) or anti-ISG-56 (red) antibodies were imaged using a confocal LSR 510 microscope at 100 × magnification. All qPCR assays were analyzed by a two-tailed Student’s t-test (* denotes p < 0.05, and ns denotes non significant, ± 1 SEM). qPCR assays were performed three times and the immunoblotting assays were performed two times.

Article Snippet: After a 1 hr blocking step at RT with 2% normal goat serum containing 0.4% Triton-X, neurons were probed with mouse monoclonal-anti-WNV-E (1:50, Abcam) and rabbit polyclonal anti-ISG-56 antibodies (1:100) overnight at 4°C.

Techniques: Expressing, Infection, Western Blot, Isolation, Cell Culture, In Vitro, Microscopy, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: (A) WT and Tlr8−/− mice brains were isolated at day 6 p.i. and imaged to detect cellular apoptosis in midsagittal brain sections. TUNEL labeling (green, white arrow) and DAPI (blue, dashed white arrow) were merged indicating reduced TUNEL immunofluorescence was observed in Tlr8−/− Purkinjie neurons of the cerebellum compared to WT controls (n = 4 per group). Gene expression profile of Bax (B) to β-actin in different brain regions: Cerebellum, Cortex (Ctx), Midbrain (Mid), Olfactory Bulb (OB), and spinal cords (SC) from WNV-infected WT and Tlr8−/− mice (n = 9-22 per group) at day 4 p.i. were analyzed by qPCR. Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6 to 12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr. Gene expression of Bax (C) to β-actin was analyzed by qPCR. Gene expression of Isg-56 (D), WNV-E (E) and Bax (F) were measured by qPCR in Neuro-2a cells transfected with siRNA targeting Isg-56 (10 nM) for 24 hr, followed by infection with WNV (MOI = 5) for an additional 48 hr. (G and H) Neuro-2a cells were transfected with siRNA targeting Isg-56 or with a scrambled siRNA control and infected with WNV, as above. Cells were stained with annexin V and PI followed by flow cytometric analysis (n = 3 per group). Brain sections were imaged using a confocal LSR 510 microscope at 63 × magnification. All qPCR and flow cytometric analyses were performed three times and analyzed using a two-tailed Student’s t-test (* denotes p < 0.05, ± 1 SEM).

Article Snippet: After a 1 hr blocking step at RT with 2% normal goat serum containing 0.4% Triton-X, neurons were probed with mouse monoclonal-anti-WNV-E (1:50, Abcam) and rabbit polyclonal anti-ISG-56 antibodies (1:100) overnight at 4°C.

Techniques: Isolation, TUNEL Assay, Labeling, Immunofluorescence, Expressing, Infection, Cell Culture, In Vitro, Transfection, Staining, Microscopy, Two Tailed Test

Journal: Experimental Biology and Medicine

Article Title: TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

doi: 10.3389/ebm.2024.10122

Figure Lengend Snippet: Expression of ISG56 in cultured human RFLS incubated with poly I:C (A) RFLS were cultured and treated with 0.4–50 μg/mL poly I:C. After 8 h of incubation, RNA was extracted, cDNA was synthesized, and quantitative real-time PCR was performed to amplify ISG56 and GAPDH cDNA. ISG56 mRNA expression was normalized to that of GAPDH. Data for ISG56 are shown as fold increase relative to that of unstimulated cells. (B) Cells were treated with poly I:C and lysed 16 h post-incubation. The lysates were subjected to western blotting to determine ISG56 and actin protein expression. (C,D) Time course of ISG56 mRNA and protein expression in RFLS incubated with poly I:C. The cells were treated with 30 μg/mL poly I:C for up to 24 h, and RNA and protein were obtained. ISG56 mRNA and protein expression was measured using qRT-PCR (C) and western blotting (D) , respectively. Data in (A,C) represent the mean ± standard deviation ( n = 3). * p < 0.01, by Student’s t-test.

Article Snippet: The membranes were blocked and incubated, with rabbit antibodies against TLR3 (Cell Signaling Technology, Danvers, MA, United States; 1:1000 dilution), IFIT1/ISG56 (Genetex, Irvine, CA, United States; 1:2500 dilution), MDA5 (Immuno-Biological Laboratories, Gunma, Japan; 1:2000 dilution), or actin (Sigma-Aldrich; 1:5,000 dilution), overnight at 4°C.

Techniques: Expressing, Cell Culture, Incubation, Synthesized, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

doi: 10.3389/ebm.2024.10122

Figure Lengend Snippet: Expression of ISG56 under treatment with a neutralizing type I IFN antibody mixture in RFLS incubated with poly I:C Cells were preincubated with human type I IFN-neutralizing antibody mixture (1:25 dilution) for 1 h and further treated with 30 μg/mL poly I:C for 16 h. (A) RNA was extracted and qRT-PCR was performed. (B) Cells were lysed and ISG56 and actin protein expression were determined using western blotting. Data in (A) represent the mean ± standard deviation ( n = 3). * p < 0.01, by Student’s t-test.

Article Snippet: The membranes were blocked and incubated, with rabbit antibodies against TLR3 (Cell Signaling Technology, Danvers, MA, United States; 1:1000 dilution), IFIT1/ISG56 (Genetex, Irvine, CA, United States; 1:2500 dilution), MDA5 (Immuno-Biological Laboratories, Gunma, Japan; 1:2000 dilution), or actin (Sigma-Aldrich; 1:5,000 dilution), overnight at 4°C.

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

doi: 10.3389/ebm.2024.10122

Figure Lengend Snippet: Expression of CXCL10 and MDA5 in poly IC-treated RFLS under the introduction of small interfering RNA (siRNA) against ISG56 Cells were transfected with siRNA against ISG56 and incubated for 48 h. Next, cells were treated with 30 μg/mL poly I:C for another 16 h. RNA was extracted and qRT-PCR was performed to determine CXCL10 (A) , MDA5 (B) , and ISG56 (C) mRNA expression. (D) The medium was collected and subjected to CXCL10 ELISA. Data from (A–D) represent the mean ± standard deviation ( n = 3). * p < 0.01 by Student’s t-test. NS, not significant by Student’s t-test.

Article Snippet: The membranes were blocked and incubated, with rabbit antibodies against TLR3 (Cell Signaling Technology, Danvers, MA, United States; 1:1000 dilution), IFIT1/ISG56 (Genetex, Irvine, CA, United States; 1:2500 dilution), MDA5 (Immuno-Biological Laboratories, Gunma, Japan; 1:2000 dilution), or actin (Sigma-Aldrich; 1:5,000 dilution), overnight at 4°C.

Techniques: Expressing, Small Interfering RNA, Transfection, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

doi: 10.3389/ebm.2024.10122

Figure Lengend Snippet: Expression of ISG56 under treatment with siRNA against MDA5 in RFLS Cells were transfected with siRNA against MDA5 and incubated for 48 h. Next, cells were treated with 30 μg/mL poly I :C for 16 h. RNA was extracted and qRT-PCR was performed to determine ISG56 (A) and MDA5 (C) mRNA expression. (B) Cells were lysed; ISG56, MDA5 and actin protein expression were measured using western blotting. Data from (A , C) represent the mean ± standard deviation ( n = 3). * p < 0.01, by Student’s t-test.

Article Snippet: The membranes were blocked and incubated, with rabbit antibodies against TLR3 (Cell Signaling Technology, Danvers, MA, United States; 1:1000 dilution), IFIT1/ISG56 (Genetex, Irvine, CA, United States; 1:2500 dilution), MDA5 (Immuno-Biological Laboratories, Gunma, Japan; 1:2000 dilution), or actin (Sigma-Aldrich; 1:5,000 dilution), overnight at 4°C.

Techniques: Expressing, Transfection, Incubation, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Experimental Biology and Medicine

Article Title: TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

doi: 10.3389/ebm.2024.10122

Figure Lengend Snippet: Immunohistochemical analysis for ISG56 in knee synovial tissue from osteoarthritis and rheumatoid arthritis patients Synovial tissue was collected from patients that underwent total knee arthroplasty. This included patients with osteoarthritis (OA) [cases 1-4, n = 4, (A) ] and rheumatoid arthritis (RA) [cases 5-8, n = 4, (B) ]. ISG56 expression was examined by immunohistochemistry. The immunoreactivity of ISG56 was stronger in RA synovial cells than in OA synovial cells.

Article Snippet: The membranes were blocked and incubated, with rabbit antibodies against TLR3 (Cell Signaling Technology, Danvers, MA, United States; 1:1000 dilution), IFIT1/ISG56 (Genetex, Irvine, CA, United States; 1:2500 dilution), MDA5 (Immuno-Biological Laboratories, Gunma, Japan; 1:2000 dilution), or actin (Sigma-Aldrich; 1:5,000 dilution), overnight at 4°C.

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry

Journal: Experimental Biology and Medicine

Article Title: TLR3 signaling-induced interferon-stimulated gene 56 plays a role in the pathogenesis of rheumatoid arthritis

doi: 10.3389/ebm.2024.10122

Figure Lengend Snippet: Predicted role of ISG56 expression via TLR3 in RFLS TLR3 activation by poly I:C induces the expression of CXCL10 in RFLS. CXCL10 induction is positively regulated by ISG56, and ISG56 expression is upregulated by type I IFN and positively regulated by MDA5. CXCL10 induces lymphocyte chemotaxis followed by synovial inflammation. Thus, ISG56 may be involved in rheumatoid synovial inflammation, at least in part, by enhancing TLR3-mediated CXCL10 expression.

Article Snippet: The membranes were blocked and incubated, with rabbit antibodies against TLR3 (Cell Signaling Technology, Danvers, MA, United States; 1:1000 dilution), IFIT1/ISG56 (Genetex, Irvine, CA, United States; 1:2500 dilution), MDA5 (Immuno-Biological Laboratories, Gunma, Japan; 1:2000 dilution), or actin (Sigma-Aldrich; 1:5,000 dilution), overnight at 4°C.

Techniques: Expressing, Activation Assay, Chemotaxis Assay

Journal: Journal of Virology

Article Title: Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System

doi: 10.1128/JVI.00856-16

Figure Lengend Snippet: NeoB reduced lipid accumulation in hepatocyte-derived cell lines. (A) Oil red O staining was performed as described in Materials and Methods with Huh7.5.1 and Huh7-25 cells treated with 0.2% DMSO (control; a and e), 20 μM NeoB (b and f), 20 μM NeoA (c and g), or 30 μM 5CPPSS-50 (d and h) for 72 h. (B) mRNAs for SREBP1c and GAPDH were detected in Huh-7.5.1 cells treated with or without the indicated compounds in the presence of 5 μM TO-901317 for 24 h. (C) Uncleaved and cleaved forms of SREBP1 and actin proteins were detected by immunoblotting using Huh-7 cells treated with or without NeoB (5, 10, and 20 μM) as described in Materials and Methods. The arrow indicates the cleaved SREBP1 protein. The values below the panels indicate the band intensities of cleaved SREBP1 and actin proteins. (D and E) Oil red O staining was performed on HCV-infected Huh7 cells or primary human hepatocytes treated with 0.2% DMSO (control; i, i-2, and l), 20 μM NeoB (j, j-2, and m), or 20 μM NeoA (k, k-2, and n) for 72 h. Panels i-2, j-2, and k-2 are higher-magnification images of the boxed areas shown in panels i, j, and k, respectively.

Article Snippet: Mouse anti-HCV core protein (2H9), rabbit anti-NS5A, rabbit anti-ISG56 (Abcam), rabbit anti-MxA (Santa Cruz Biotechnology), anti-LXR (Abcam), rabbit anti-SCD-1 (Cell Signaling Technology), mouse anti-SREBP1 (BD Pharmingen), and mouse anti-β-actin (Sigma) antibodies were used as primary antibodies.

Techniques: Derivative Assay, Staining, Western Blot, Infection