Journal: Frontiers in Immunology

Article Title: High Expression of IL-36γ in Influenza Patients Regulates Interferon Signaling Pathway and Causes Programmed Cell Death During Influenza Virus Infection

doi: 10.3389/fimmu.2020.552606

Figure Lengend Snippet: IL-36γ enhance IFNs production and promote IAV-mediated IFN-stimulated STAT1 phosphorylated inhibition in lung epithelial cells. (A) A549 cells were treated with IL-36γ (100 ng/ml) or left untreated 24 h prior to infection with IAV-C07 (MOI = 1). Levels of IFN-α, IFN-β, and IFN-λ1 in supernatant were measured by ELISA in different timeframes, as indicated. (B) Experiments performed as in (A) , except levels of IFN-stimulated genes (ISGs), such as OSA, PKR, and Mx1, were quantified using RT-PCR. (C) Simple medium only and IL-36γ pre-treated A549 cells were further infected with IAV-Ca07 (MOI = 1), then cells were collected and lysed at the indicated time, and the levels of p-STAT1 and p-STAT2, non-phosphorylated STAT1 and STAT2, Mx1, PKR, IAV-Ca07 NP, and M1 proteins were assessed by western blotting and Image J. (D) A549 cells were stimulated by supernatant culture medium from IAV-infected cells in (C) , followed by western blotting with indicated antibodies. (E, F) Experiments performed as in (C, D) , except 16HBE cells used. The data were shown as mean ± SD. Statistical significance is determined by Student’s t test. *P < 0.05, **P < 0.01.

Article Snippet: The following reagents were used in western blotting: antibodies against human IL-36γ, LC-3B, p62, Mx1, PKR (Abcam), phosphorylated (p)-STAT1 (pY701), STAT1, (p)-STAT2 (pY141), STAT2, p-Akt, Akt, p-mTOR, mTOR, p-ULK1, ULK1, and GAPDH (Cell Signal Technology) and antibodies against IAV-Nucleocapsid (NP) and Matrix protein 1 (M1) (Sino Biological).

Techniques: Inhibition, Infection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

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: Frontiers in immunology

Article Title: High Expression of IL-36γ in Influenza Patients Regulates Interferon Signaling Pathway and Causes Programmed Cell Death During Influenza Virus Infection.

doi: 10.3389/fimmu.2020.552606

Figure Lengend Snippet: FIGURE 7 | IL-36g promote apoptosis and inhibit autophagy in IAV-infected lung epithelial cells. (A) A549 cells were infected with or without IAV-Ca07 (MOI = 1) and treatment with or without IL-36g, and apoptotic cells were determined by flow cytometry. The early apoptosis ratio and total apoptosis ratio was analyzed using Flow J software. (B) Western blotting analysis of endogenous LC-3B and p62 protein in A549 cells upon infected with or without IAV-Ca07 and treatment with or without IL-36g at indicated time points. (C) Western blotting analysis of Akt, mTOR, ULK1, and LC-3B in A549 cells infected with IAV-Ca07 and treatment with IL- 36g (100 ng/ml) for 6 h and 12 h. (D) The relative expression of Akt, mTOR, ULK1, and LC-3B was consistent with that shown in (C). (E) Transmission electron microscopy (TEM) analysis showing the formation of autophagosomes in A549 cells after infected with or without IAV-Ca07 and treatment with or without IL-36g for 12 h. Representative autophagosomes or autolysosomes (black arrows) in A549 cell are highlighted in the enlarged images (right). The data were shown as mean ± SD. Statistical significance is determined by Student’s t test. *P < 0.05, **P < 0.01.

Article Snippet: The following reagents were used in western blotting: antibodies against human IL-36g, LC-3B, p62, Mx1, PKR (Abcam), phosphorylated (p)-STAT1 (pY701), STAT1, (p)STAT2 (pY141), STAT2, p-Akt, Akt, p-mTOR, mTOR, pULK1, ULK1, and GAPDH (Cell Signal Technology) and antibodies against IAV-Nucleocapsid (NP) and Matrix protein 1 (M1) (Sino Biological).

Techniques: Infection, Cytometry, Software, Western Blot, Expressing, Transmission Assay, Electron Microscopy

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: Oncology Research

Article Title: Downregulation of MicroRNA-135 Promotes Sensitivity of Non-Small Cell Lung Cancer to Gefitinib by Targeting TRIM16

doi: 10.3727/096504017X15144755633680

Figure Lengend Snippet: Alteration of the JAK/STAT signaling pathway after aberrant expression of miR-135 and TRIM16 in NSCLC cells. Phosphorylated levels of key kinases involved in the JAK/STAT pathway in H1650 cells (A) and H1975 cells (B) were measured by Western blot analysis under different transfection conditions as described in the figure. JAK1, Janus-activated kinase-1; STAT, signal transducer and activator of transcription.

Article Snippet: Then proteins in the gels were transferred to polyvinylidene difluoride (PVDF) membranes, and the membranes were blocked by 5% nonfat milk, followed by incubation at 4°C overnight with primary antibodies against B-cell lymphoma-2 (Bcl-2; ab194583), Bcl-2-associated X protein (Bax; ab53154), procaspase 3 (ab90437), cleaved caspase 3 (ab2302), procaspase 9 (ab2013), cleaved caspase 9 (ab2324), E-cadherin (ab133597), β-catenin (ab6302), programmed death ligand 1 (PD-L1; ab213524), TRIM16 (ab72129), GAPDH (ab181603) (all from Abcam, Cambridge, UK), Janus-activated kinase-1 (JAK1; 3332), phosphorylated JAK1 (p-JAK1; 3331), signal transducer and activator of transcription (STAT) 1 (9176), phosphorylated STAT1 (p-STAT1; 9177), STAT2 (4594), or phosphorylated STAT2 (p-STAT2; 4441) (all from Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Western Blot, Transfection

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