Journal: Journal of Virology

Article Title: Selective Interferon Responses of Intestinal Epithelial Cells Minimize Tumor Necrosis Factor Alpha Cytotoxicity

doi: 10.1128/JVI.00603-20

Figure Lengend Snippet: IECs in the neonatal intestine are minimally responsive to IFN-β. Neonatal mice were injected with either IFN-β or IFN-λ3 for 4 h. (A) Small intestinal tissue was isolated and stained with DAPI and Usp18 antisense probes. The dashed line indicates the approximate boundary between villi and the lamina propria (LP). (B) Usp18 abundance in sorted IECs (EpCAM positive/CD45 negative) and hematopoietic cells (CD45 positive/EpCAM negative) was determined by qPCR. (C) Mice were inoculated with rotavirus (RV), and viral genomes were quantitated in intestines 20 h later. (D) Comparison of IFN receptor gene abundances by qPCR in sorted EpCAM+ IECs and CD45+ hematopoietic cells. (E) Flow cytometry staining of IFNAR1 and IFNAR2 on EpCAM+ IECs and CD45+ hematopoietic cells. Gray histograms are control stains (no IFNAR antibody), and bar graphs show geometric mean fluorescence intensities (MFI) of IFNAR fluorescence normalized to the control for each replicate. Data are combined from at least two experiments with a total of three to five mice under each experimental condition; data points indicate individual animals, with bars indicating the means. Significance was determined by one-way or two-way analysis of variance (ANOVA) (B to D) or a t test (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant.

Article Snippet: A total of 0.2 μg IFN-β (catalog number 12405-1; PBL) or IFN-λ3 (catalog number 12820-1; PBL) was administered to 7-day-old neonatal mice via subcutaneous injection; an equal volume of a diluent (PBS) was administered to littermate control mice.

Techniques: Injection, Isolation, Staining, Comparison, Flow Cytometry, Control, Fluorescence

Journal: Journal of Virology

Article Title: Selective Interferon Responses of Intestinal Epithelial Cells Minimize Tumor Necrosis Factor Alpha Cytotoxicity

doi: 10.1128/JVI.00603-20

Figure Lengend Snippet: IEC organoids are dually responsive to IFN-β and IFN-λ. (A) Representative images of IEC organoids. (B) IEC organoids were treated with the indicated concentrations of recombinant IFN-β or IFN-λ2 for the indicated times, and RNA was isolated for the quantitation of Isg15, Usp18, and Cxcl10 transcripts by qPCR. The dotted line indicates the limit of detection. (C) Transcript abundances of the indicated genes 4 h after IFN treatment across the range of IFN doses tested. Dashed lines indicate maximal responses for IFN-β or IFN-λ. (D) IEC organoids pretreated with PBS, IFN-β, or IFN-λ3 for 8 h were infected with rotavirus. Viral genomes were quantified at the indicated times postinfection. Data are from two (B and C) or three (D) experiments with duplicate treatment/infection wells; error bars show standard errors of the means (SEM). Statistical significance was determined by a t test (C) or one-way ANOVA (D). *, P < 0.05; **, P < 0.01.

Article Snippet: A total of 0.2 μg IFN-β (catalog number 12405-1; PBL) or IFN-λ3 (catalog number 12820-1; PBL) was administered to 7-day-old neonatal mice via subcutaneous injection; an equal volume of a diluent (PBS) was administered to littermate control mice.

Techniques: Recombinant, Isolation, Quantitation Assay, Infection

Journal: Journal of Virology

Article Title: Selective Interferon Responses of Intestinal Epithelial Cells Minimize Tumor Necrosis Factor Alpha Cytotoxicity

doi: 10.1128/JVI.00603-20

Figure Lengend Snippet: IFN-β-specific apoptosis ISGs are dependent on the canonical transcription factor STAT1. (A) Reanalysis of ChIP-seq data under GEO series accession number GSE115433 (52). Percentages of genes in common ISG and IFN-β-specific ISG sets that had significant ChIP-seq peaks for STAT1, STAT2, or IRF9 within 500 bp of the transcription start site are shown. Statistical significance was determined by a chi-square test of contingency tables of genes with or without peaks. *, P < 0.05; ****, P < 0.0001. (B and C) Quantitative PCR analysis of genes indicated on the x axis following treatment of WT or Stat1−/− IEC organoids with 10 ng/ml IFN-β (B) or IFN-λ3 (C) for 4 h, normalized to PBS-treated controls. Data are combined from four experiments, with * indicating a P value of <0.05 by one-way ANOVA.

Article Snippet: A total of 0.2 μg IFN-β (catalog number 12405-1; PBL) or IFN-λ3 (catalog number 12820-1; PBL) was administered to 7-day-old neonatal mice via subcutaneous injection; an equal volume of a diluent (PBS) was administered to littermate control mice.

Techniques: ChIP-sequencing, Real-time Polymerase Chain Reaction

Journal: Journal of Virology

Article Title: Selective Interferon Responses of Intestinal Epithelial Cells Minimize Tumor Necrosis Factor Alpha Cytotoxicity

doi: 10.1128/JVI.00603-20

Figure Lengend Snippet: IFN-β potentiates TNF-α-triggered apoptosis. (A) MTT viability assay of IEC organoids treated with 10 ng/ml IFN-λ3, IFN-β, or PBS for 4 h followed by treatment with 100 ng/ml TNF-α for 20 h. (B and C) Cleaved caspase 3 (CC3) in IEC organoids was assessed by immunofluorescence following pretreatment with 10 ng/ml IFN-λ3, IFN-β, or PBS for 4 to 8 h and subsequent treatment with medium or 100 ng/ml TNF-α for 16 to 20 h. The positive apoptosis control, staurosporine (SS), was administered to PBS organoids for 16 to 20 h. Data are pooled from three independent experiments, with statistical significance determined by one-way ANOVA in panel A and by a Kruskal-Wallis test with Dunn’s multiple comparisons in panel B. The solid line depicts the mean in panel A and the median in panel B. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant (P > 0.05). E-cad., E-cadherin.

Article Snippet: A total of 0.2 μg IFN-β (catalog number 12405-1; PBL) or IFN-λ3 (catalog number 12820-1; PBL) was administered to 7-day-old neonatal mice via subcutaneous injection; an equal volume of a diluent (PBS) was administered to littermate control mice.

Techniques: MTT Viability Assay, Immunofluorescence, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Human blood dendritic cell antigen 3 (BDCA3)(+) dendritic cells are a potent producer of interferon-λ in response to hepatitis C virus.

doi: 10.1002/hep.26182

Figure Lengend Snippet: Fig. 3. BDCA3þ DCs recov- ered from peripheral blood or intrahepatic lymphocytes produce large amounts of IL-29/IFN-k1, IL-28A/IFN-k2, and IL-28B/IFN- k3 in response to poly IC. (A) BDCA3þ DCs and mDCs were cultured at 2.5 104 cells with 25 mg/mL poly IC, and pDCs were with 5 mM CPG for 24 hours. The supernatants were examined for IL-29, IL-28A, IL-28B, IFN-b and IFN-a. Results are shown as mean 6 SEM from 15 experiments. *P < 0.05; ***P < 0.0005 by Krus- kal-Wallis test. (B) For the IL-28B production, BDCA3þ DCs in intrahepatic lymphocytes were cultured at 2.5 104 cells with 25 mg/mL poly IC for 24 hours. The samples of cases 8 and 9 were obtained from patients with non-B, non-C liver disease and that of case 17 was from an HCV-infected patient (Supporting Table 1).

Article Snippet: IL-28B/IFN-k3 was quantified by a newly developed chemiluminescence enzyme immu- noassay (CLEIA) system.15 IL-29/IFN-k1, IL-28A/ IFN-k2, and IFN-b were assayed by commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, R&D, and PBL, respectively).

Techniques: Cell Culture, Infection

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 permits hepatocyte-specific expression of IFNλ3. HepG2.2.15, HEK293 and Hela cells were transfected with MC vectors. a Schematic illustration of the MC.IFNs. MC.IFNα is 1656-bp in length, MC.IFNλ3 is 1677-bp in length. attR represents a 36-bp attR recombinant site. ApoE indicates ApoE promoter. CDS represents coding sequence. bpA represents bovine growth hormone polyadenylation signal. b The expression of IFNα and IFNλ3 in cell lysate was determined by Western Blot at 3 days post-transfection. Lane 1–5 represents the untreated control (HepG2.2.15 cells without MC transfection), MC.IFNα transfected HepG2.2.15 cells, and MC.IFNλ3 transfected HepG2.2.15 cells, MC.IFNλ3 transfected HEK293 cells, MC.IFNλ3 transfected Hela cells, respectively

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Expressing, Transfection, Recombinant, Sequencing, Western Blot, Control

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 inhibits viral antigens expression and viral DNA replication in HepG2.2.15 cells. HepG2.2.15 cells were transfected with MC.IFNλ3 and MC.IFNα. While the untreated HepG2.2.15 cells served as a blank control (Blank). The levels of viral antigens, namely HBsAg ( a ) and HBeAg ( b ), and viral DNA in cell culture supernatant were determined by chemiluminiscence and qPCR, respectively, at the indicated time-points (3 or 6 days post-transfection). All data are shown as mean ± SD from three independent experiments. * indicates statistically significant ( P -value < 0.05), ns indicates not significant ( P -value > 0.05)

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Expressing, Transfection, Control, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: Viral antigens and viral DNA in HepG2.2.15 cell culture supernatant after transfection

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Cell Culture, Transfection, Control

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 induce JAK1 and STAT1/STAT2 phosphorylation in HepG2.2.15 cells. HepG2.2.15 cells were transfected with MC vectors. The levels of a STAT1/STAT2 proteins and their phosphorylated form (p-STAT1/p-STAT2), b JAK1 and phosphorylated JAK1 (p-JAK1) in transfected HepG2.2.15 cells were determined by Western Blot at 6 days post-transfection. Lane 1, 2 and 3 represents untreated Control, MC.IFNα, and MC.IFNλ3 group, respectively

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Phospho-proteomics, Transfection, Western Blot, Control

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 up-regulates ISGs expression in HepG2.2.15 cells. MC.IFNλ3 up-regulates ISGs expression in HepG2.2.15 cells. The relative mRNA transcriptional levels of ten ISGs MC transfected HepG2.2.15 cells were quantified at 3 or 6 days post-transfection by qPCR. The ISGs mRNA levels in HepG2.2.15 cells after MC.IFNλ3 ( a ) and MC.IFNα ( b ) treatment were compared between 3 days and 6 days post-transfection groups. The ISGs mRNA levels in HepG2.2.15 cells between MC.IFNλ3 and MC.IFNα treatment groups were compared at 3 days ( c ) or 6 days ( d ) post-transfection. All data are shown as mean ± SD from three independent experiments

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Expressing, Transfection