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: American Journal of Physiology - Cell Physiology

Article Title: JAKinibs prevent persistent, IFNγ-autonomous endothelial cell inflammation and immunogenicity

doi: 10.1152/ajpcell.00298.2022

Figure Lengend Snippet: Interferon (IFNγ)-induced STAT1 transcriptional activity and Janus kinase (JAK)/STAT activation is persistent after cytokine withdrawal. A : HEK-Dual IFNγ/STAT1 reporter cells were treated in duplicate wells with IFNγ continuously, or with IFNγ for 3 h followed by normal medium without cytokine. After 24 h, secreted alkaline phosphatase (SEAP) reporter was measured in the medium. Results show mean STAT1 reporter signal from two technical replicates, representative of three independent experiments. B : primary human aortic endothelial cells were treated with IFNγ continuously, or primed for 3 h followed by withdrawal. Cell lysates were probed for phosphorylation of JAK and SHP proteins by blot array. The signal intensity for each spot was calculated, and results are presented as fold increase over the internal negative control spot, from duplicate spots representative of three biological replicates. C : as in B , phosphorylation of STAT proteins was measured in endothelial cell lysates. D : endothelial cells were stimulated with IFNγ for 3, 6, 18, or 24 h; or stimulated with IFNγ for 3 h and rested in normal medium for an additional 3, 15, or 21 h before cell lysates were collected. mRNA was measured by Nanostring. Heat map shows relative JAK, STAT, and IRF gene expression, using hierarchical clustering by Pearson’s minus one correlation. E : discrete normalized mRNA counts for JAK, STAT, and interferon response genes with persistent expression are presented. The longitudinal levels of each transcript are shown under continuous stimulation (black circles, bold lines) or after prime/rest (open circles, dashed lines). F : discrete normalized mRNA counts for JAK, STAT, and interferon response genes with declining expression are presented. The longitudinal levels of each transcript are shown under continuous stimulation (black circles, bold lines) or after prime/rest (open circles, dashed lines). IRF, interferon regulatory protein 1; SHP, SH2 domain-containing protein tyrosine phosphatase 2; SOCS, suppressors of cytokine signaling.

Article Snippet: Phosphorylation of intracellular signaling proteins was measured using the Human JAK/STAT Pathway Phosphorylation Array (RayBiotech).

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Negative Control, Gene Expression, Expressing

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: 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