Journal: Scientific Reports

Article Title: An orally available, small-molecule interferon inhibits viral replication

doi: 10.1038/srep00259

Figure Lengend Snippet: (a, b) The anti-HCV replicon activity of RO8191 was attenuated by knockdown of IFNAR2 (b), but not IFNAR1 (a). Inhibition of HCV replicon replication by each agent is shown (the mean and SD from 3 experiments). The HCV replicon cells were transfected with 50 nM of the indicated siRNAs (blue, red, and yellow bars). Forty-eight hours after transfection, the HCV replicon cells were treated with 1.5 μM RO8191 or 3 IU/mL IFN-α for 24 h. Twenty-four hours after treatment with each agent, the replication levels of HCV RNA were analyzed using a luciferase assay. (c, d) U5A cells that lack IFNAR2 were transfected with either an empty vector or a vector expressing the IFNAR2 gene. (c) Forty-eight hours after transfection, the cells were treated with 50 μM RO8191 (red bars) or 100 IU/mL IFN-α (yellow bars). After an additional 8 h of incubation, total RNA was extracted from the U5A cells, and the OAS1 mRNA level was measured using real-time RT-PCR. The values shown are relative to the mRNA level of human β-actin . (d) Forty-eight hours after transfection, the cells were lysed, and the whole cell lysates were immunoblotted with the indicated antibodies. (e) Real-time kinetic SPR analysis of the binding of RO8191 to the IFNAR2 ECD (red and blue lines). The results are consistent with 1:1 binding. PEG-IFN-α2a was also injected as a positive interacting control for IFNAR2 (black line, K D : 30 nM). (f, g) The phosphorylation of STAT1 was attenuated by a knockdown of IFNAR2 (g) but not IFNAR1 (f). The HCV replicon cells were transfected with the indicated siRNAs (10 nM). Forty-eight hours after transfection, the cells were treated for 15 min with 10 μM RO8191 or 200 IU/mL IFN-α. The total lysates were subjected to western blot analysis to analyze the phosphorylated and total protein levels of STAT1. The data were statistically analyzed using Student's t -test.

Article Snippet: Anti-IFNAR1 mouse monoclonal (MAB245) and anti-IFNAR2 sheep polyclonal antibodies were purchased from R&D Systems.

Techniques: Activity Assay, Knockdown, Inhibition, Transfection, Luciferase, Plasmid Preparation, Expressing, Incubation, Quantitative RT-PCR, Binding Assay, Injection, Control, Phospho-proteomics, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics

doi: 10.15252/emmm.202012924

Figure Lengend Snippet: IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO‐110 treatment (0.5 µg/ml) of SK‐Mel‐147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Quantification of the impact of BO‐110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK‐Mel‐147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate. BO‐110‐driven blockade of the tube‐forming capacity of HLEC and rescue with anti‐IFNAR1 blocking antibodies. Images correspond to cells plated in Matrigel and imaged 8 h after treatment with 0.5 µg/ml BO‐110. See also Fig , for additional results with anti‐IFNAR1 and anti‐IFN‐β blocking antibodies. Growth of B16 melanoma xenografts in siblings of Ifnar1 +/+ , Ifnar1 +/− , or Ifnar1 −/− mice. Treatment started 10 days after tumor cell implantation. BO‐110 was administered at 0.8 mg/kg, every third day for 2 weeks. N = 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by two‐way ANOVA. Histological analyses of lymphatic vessel density by Lyve1 (blue) and Prox1 (purple) in representative lymph nodes of animals in (D) processed at the endpoint of the experiment (four doses of BO‐110 or vehicle control). N = 6 mice per experimental condition. See Fig for a more complete view of these lymph nodes, where dual Prox1‐Lyve1‐positive cells were pseudocolored in red.

Article Snippet: For type I interferon blocking assays, human IFN‐β blocking antibody (Clone AF814; R&D) and its corresponding isotype (Goat IgG, R&D Systems) were used at a concentration of 0.2 µg/ml.

Techniques: Blocking Assay

Journal: EMBO Molecular Medicine

Article Title: Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics

doi: 10.15252/emmm.202012924

Figure Lengend Snippet: Binding sites for experimentally validated IRF‐related transcription factors in the promoters of MDK and FLT4 ( VEGFR3 ). Data from Interferome and TRANSFAC ( www.genexplain.com ). BO‐110‐driven blockade of the tube‐forming capacity of HLEC and rescue with anti‐IFNAR1 blocking antibodies (anti‐IFN‐β and anti‐INFAR1) or corresponding controls. Images correspond to cells plated in Matrigel and imaged 8 h after treatment with 0.5 µg/ml BO‐110. Histological analyses of lymphatic vessel density defined by dual staining for Lyve1 (blue) and Prox1 (purple). To facilitate the visualization, cells positive for both markers were pseudocolored in red. Large magnifications of the doted squared areas can be found in Fig . Quantification of Lyve1/Prox1 staining in lymph nodes of animals in Fig , processed at the endpoint of the experiment (4 doses of BO‐110 or vehicle control). Data correspond to the mean ± SD of four biological replicates. Statistical significance was determined by the t ‐test. Source data are available online for this figure.

Article Snippet: For type I interferon blocking assays, human IFN‐β blocking antibody (Clone AF814; R&D) and its corresponding isotype (Goat IgG, R&D Systems) were used at a concentration of 0.2 µg/ml.

Techniques: Binding Assay, Blocking Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics

doi: 10.15252/emmm.202012924

Figure Lengend Snippet: A Impact of BO‐110 on tumor‐bearing mice implanted with syngeneic B16R2L (5 × 10 5 cells). When tumors have an average size on 100 mm 3 , mice were randomized into two groups and treated with BO‐110 (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points. B Quantification of Vegfr3 Luc emission in the tumor area in mice in experiment (A). C Vegfr3 Luc emission in the inguinal and brachial lymph nodes of mice in experiment (A). D–F Quantification of Vegfr3 Luc emission in the spleen, liver, and lung of mice in experiment (A), respectively. G ELISA analysis of Mdk blood levels in mice in experiment (A). H ELISA analysis of mouse Ifn‐β blood levels in mice in experiment (A). I Antitumoral effect of BO‐110 on xenografts of SK‐Mel‐147 (1 × 10 6 cells) implanted in the back of Vegfr3 Luc; nu / nu nude mice. When tumors had an average size on 150 mm 3 , mice were randomized into two groups and treated with BO‐110 (BO, 0.8 mg/kg) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points, and tumor volume was calculated as indicated in Materials and Methods. J Quantification of Vegfr3 Luc emission in the tumor area in mice in experiment (I). K–N Quantification of Vegfr3 Luc emission in the lymph nodes, spleen, liver, and lung of mice in experiment (I), respectively. O ELISA analysis of Mdk blood levels in mice from (I). P ELISA analysis of mouse Ifn‐β blood levels in mice from (I). Data information: For all panels in this figure, N = 4 mice per condition. Statistical significance was determined by two‐way ANOVA.

Article Snippet: For type I interferon blocking assays, human IFN‐β blocking antibody (Clone AF814; R&D) and its corresponding isotype (Goat IgG, R&D Systems) were used at a concentration of 0.2 µg/ml.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

doi: 10.1371/journal.ppat.1002016

Figure Lengend Snippet: TetR and TetR-IE1 cells were treated with doxycycline for 72 h and with solvent (w/o), IFN-β or IFN-γ for 24 h. Doxycycline and IFN treatment was performed in the continuous presence of normal goat immunoglobulin G (IgG), goat anti-IFN-β or goat anti-IFN-γ antibodies. Relative mRNA expression levels were determined by qRT-PCR with primers specific for the CXCL10, CXCL11, GBP4, and IE1 genes. Results were normalized to TUBB and mean values with standard deviations from two biological and two technical replicates are shown. Expression is shown in comparison to normal IgG-treated cells (set to 1).

Article Snippet: Neutralizing goat antibodies to human IFN-β (no. AF814) or IFN-γ (no. AF-285-NA) and normal goat IgG (no. AB-108-C) were purchased from R&D Systems and used at concentrations of 1 μg/ml (anti-IFN-β) or 2 μg/ml (anti-IFN-γ, normal IgG).

Techniques: Solvent, Expressing, Quantitative RT-PCR, Comparison

Journal:

Article Title: Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

doi: 10.1097/CJI.0b013e3181fb045d

Figure Lengend Snippet: Viable cell suspensions from tumor-free nodes (TFN), melanoma-infiltrated (MIN), and sentinel immunized nodes (SIN) were plated in 48 well plates at 5×105 mononuclear inflammatory cells per mL and incubated with media alone, 1×105 IU of IFN-α, or 1×105 IU of IFN-γ for 24 hours. The supernatants were then assessed for CXCL9-11 production by ELISA. Data are means ± standard deviation of ELISA results from one of three independent assays. *, p < 0.05 vs. untreated; +, p < 0.01 vs. untreated.

Article Snippet: Evaluation of human melanoma cells for expression of IFN receptors Melanoma cell lines were stained with antibodies MAB 245 (clone 85228, R&D Systems) for IFN-αR1, MAB 6731 (clone 92101, R&D systems) for IFN-γR1, and an isotype-matched control, and evaluated by flow cytometry.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

doi: 10.1097/CJI.0b013e3181fb045d

Figure Lengend Snippet: Melanoma cell lines were cultured in 24 well plates at 1×106 cells per mL, either without or with 1×105 IU of IFN–α or IFN–γ for 24 hours. The supernatants were assayed in triplicate by ELISA for CXCL9 (A), CXCL10 (B), and CXCL11 (C). Data are means ± standard deviation of ELISA results from one of three independent assays.

Article Snippet: Evaluation of human melanoma cells for expression of IFN receptors Melanoma cell lines were stained with antibodies MAB 245 (clone 85228, R&D Systems) for IFN-αR1, MAB 6731 (clone 92101, R&D systems) for IFN-γR1, and an isotype-matched control, and evaluated by flow cytometry.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

doi: 10.1097/CJI.0b013e3181fb045d

Figure Lengend Snippet: A and B) Expression of IFN receptors on human melanoma cell lines DM93 (A) and DM331 (B). Data are representative of staining for 16 human melanoma cell lines evaluated (VMM5; VMM12; VMM15; VMM18; VMM19; VMM39; VMM150; MV3; SKMel2; DM6; DM13; DM14; DM122; and DM281, not shown) from one of three similar experiments. C) Representative data for IFN receptor expression on melanoma (CD45neg CD11cneg CD14neg S100+) cells in MIN, from one of six MIN samples tested.

Article Snippet: Evaluation of human melanoma cells for expression of IFN receptors Melanoma cell lines were stained with antibodies MAB 245 (clone 85228, R&D Systems) for IFN-αR1, MAB 6731 (clone 92101, R&D systems) for IFN-γR1, and an isotype-matched control, and evaluated by flow cytometry.

Techniques: Expressing, Staining

Journal:

Article Title: Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

doi: 10.1097/CJI.0b013e3181fb045d

Figure Lengend Snippet: A-C) Chemokine production by whole blood samples following co-culture with melanoma supernatant and treatment with IFNs. Whole blood from a normal donor was incubated with escalating quantities of supernatant from a cultured melanoma cell line, either in media alone or with 1×105 IU of IFN–α or IFN–γ for 24h. The supernatants were assayed in triplicate by ELISA for CXCL9 (A), CXCL10 (B), and CXCL11 (C). Data are means ± standard deviation of pooled ELISA results from one of three independent assays using 6 normal donor patient samples.

Article Snippet: Evaluation of human melanoma cells for expression of IFN receptors Melanoma cell lines were stained with antibodies MAB 245 (clone 85228, R&D Systems) for IFN-αR1, MAB 6731 (clone 92101, R&D systems) for IFN-γR1, and an isotype-matched control, and evaluated by flow cytometry.

Techniques: Co-Culture Assay, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Interferons Induce CXCR3-cognate Chemokine Production by Human Metastatic Melanoma

doi: 10.1097/CJI.0b013e3181fb045d

Figure Lengend Snippet: Normal donor PBL were incubated for 24h in media or with 1×105 IU of IFN–γ, and with or without supernatant (50% of culture) from the DM93 melanoma cell line. Cells were stained for CD14 and intracellular CXCL9. A) Staining for CD14 and CXCL9 in untreated PBL. B) Staining for CD14 and CXCL9 in IFN-γ-treated PBL. C) Staining for CD14 and CXCL9 in IFN-γ-treated PBL cultured with 50% melanoma supernatant. Data are representative from one of three similar experiments.

Article Snippet: Evaluation of human melanoma cells for expression of IFN receptors Melanoma cell lines were stained with antibodies MAB 245 (clone 85228, R&D Systems) for IFN-αR1, MAB 6731 (clone 92101, R&D systems) for IFN-γR1, and an isotype-matched control, and evaluated by flow cytometry.

Techniques: Incubation, Staining, Cell Culture