Journal: Virology

Article Title: Kobuvirus VP3 protein restricts the IFN-β-triggered signaling pathway by inhibiting STAT2-IRF9 and STAT2-STAT2 complex formation.

doi: 10.1016/j.virol.2017.04.023

Figure Lengend Snippet: Fig. 1. PKV VP3 negatively regulates the IFN-β-triggered signaling pathway. (A) Overexpression of PKV proteins on IFN-β-triggered STAT/STAT2/IRF9, SeV-triggered IFN- β and IFN-γ-triggered IRF1 promoter activation. PKV protein expression was quantified with Western blotting. (B) Dose-dependent effects of PKV VP3 and L on IFN-β-triggered activation of the STAT1/STAT2/IRF9 promoter. The experiment was performed as described in A. (C) RT-qPCR analysis of HEK293 and HeLa cells transfected with a control, PKV VP3 or L for 24 h followed by IFN-β or IFN-γ (50 ng/ml) treatment for 0–8 h. (D-E) The empty vector or FLAG-VP3 stably expressing MEFs (2×106) stimulated or left unstimulated with IFN-α (20 U/ml) for 2 h and infected with VSV-GFP for 24 h, the supernatants were analyzed by the plaque assay (D) and the GFP intensities were analyzed by flow cytometry (E). Data were one representative from at least three independent experiments with three technical replicates (*p < 0.05, ***p < 0.001).

Article Snippet: Antibodies against IRF9, STAT2, and phosphorylated STAT1 (pY701-STAT1) were purchased from Cell Signaling Technology (CST, MA, USA).

Techniques: Over Expression, Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Stable Transfection, Infection, Plaque Assay, Cytometry

Journal: Virology

Article Title: Kobuvirus VP3 protein restricts the IFN-β-triggered signaling pathway by inhibiting STAT2-IRF9 and STAT2-STAT2 complex formation.

doi: 10.1016/j.virol.2017.04.023

Figure Lengend Snippet: Fig. 2. PKV VP3 affects ISGF3 complex nuclear translocation induced by IFN-β. (A) Western blotting was used to detect pY701-STAT1, STAT1, pY690-STAT2, STAT2, and FLAG-VP3 expression in HEK293 cells. (B) HEK293 cells stably expressing VP3 were treated with IFN-β (50 ng/ml) and Western blotting was used to detect pY701-STAT1, STAT1, pY690-STAT2, STAT2, IRF9 and FLAG-VP3 expression in the cytoplasm, nucleus and whole cell lysate. (C) Immunofluorescent analysis of HeLa cells transfected with IRF9, STAT1, STAT2, FLAG-VP3 and FLAG-L for 20 h followed by IFN-β (50 ng/ml) stimulation for 0–2 h. The nuclear localization rate was analyzed by Image J. Data were one representative of three independent experiments.

Article Snippet: Antibodies against IRF9, STAT2, and phosphorylated STAT1 (pY701-STAT1) were purchased from Cell Signaling Technology (CST, MA, USA).

Techniques: Translocation Assay, Western Blot, Expressing, Stable Transfection, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: A , B Cytoplasmic retention of Flag-tagged WT STAT1 in IFNγ-stimulated cells co-expressing R602L/Y701F-GFP. Transfected STAT1-negative U3A and HeLa cells expressing R602L/Y701F-GFP and WT-Flag were treated with 50 ng/ml IFNγ for 45 min and subsequently stained with an anti-Flag antibody. A The fluorescence micrographs show the intracellular distribution of Flag-tagged STAT1 co-expressed in cells transfected with R602L/Y701F-GFP as well as the localization of the corresponding Hoechst-stained nuclei (n = 3 independent transfections). B The histogram demonstrates the nucleocytoplasmic STAT1-Flag distribution in the absence and presence of R602L/Y701F-GFP in IFNγ-treated HeLa cells, as determined by the ratio of nuclear-to-total fluorescence intensity (means ± standard deviations from n = 20 cells, * p ≤ 0.05, as assessed by a two-tailed Student’s t test). C The absence of nuclear P-STAT1 staining of the endogenous protein in HeLa cells expressing R602L/Y701F-GFP. HeLa cells were transfected with R602L/Y701F-GFP and stimulated with IFNγ for indicated times before staining with an anti-phospho-tyrosine antibody. D Crystal structure of the STAT1 anti-parallel dimer. A critical arginine residue at position 274 marked in cyan in the coiled-coil domains and threonine residue at position 385 marked in pink in the DNA-binding domains of the two STAT1 proteins (white and orange). Structural data were obtained from the Protein Data Bank (pdb) file 1YVL for the STAT1 anti-parallel dimer. E The absence of nuclear P-STAT1 staining of endogenous STAT1 in HeLa cells expressing R274W/R602L/Y701F-GFP. HeLa cells were transfected with R274W/R602L/Y701F-GFP and stimulated with IFNγ for indicated times before being stained with an anti-phospho-tyrosine antibody. Scale bars in A , C , E mark a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Expressing, Transfection, Staining, Fluorescence, Two Tailed Test, Residue, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: The dimerization-deficient quadruple mutant (QM-GFP) mimicking U-STAT1 inhibits the detection of co-expressed WT protein but does not affect tyrosine phosphorylation. A – C HeLa cells were transfected with a plasmid coding for QM-GFP and stimulated with IFNγ for 45 min followed by staining with anti-phospho-tyrosine antibody. A The fluorescence micrographs show the intracellular distribution of QM-GFP and WT P-STAT1, as well as the localization of the corresponding Hoechst-stained nuclei. B Histogram demonstrating the net reduction of nuclear P-STAT1 intensity in HeLa cells expressing QM-GFP, as determined by the ratio of nuclear-to-total fluorescence intensity (n = 3 independent transfections, means ± standard deviations from n = 20 cells, * p ≤ 0.05 by a two-tailed Student’s t test). C Concentration-dependent inhibition of nuclear P-STAT1 accumulation by co-expressed QM-GFP. The fluorescence intensities of nuclear P-STAT1 staining were plotted against the total cellular QM-GFP fluorescence. D , E Representative immunoblot of whole cell extracts from STAT1-negative U3A cells co-expressing untagged STAT1 and QM-GFP after treatment for 45 min with 50 ng/ml of recombinant IFNγ and the quantification thereof from three independent transfection experiments. F , G An in vitro phosphorylation assay demonstrated no difference in tyrosine phosphorylation rates of the WT STAT1 by JAK2 with respect to the presence or absence of QM-GFP. Whole cell extracts (10 μl in each reaction) from reconstituted U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP were incubated with 4 μg/ml of recombinant JAK2 kinase and the levels of P-STAT1 were monitored over time by means of Western blotting (n = 3). Statistical analysis revealed no significant difference in the phosphorylation kinetics of the WT in the presence of either QM-GFP or WT-GFP. H , I Results from an in vitro dephosphorylation assay using extracts from IFNγ-pre-stimulated U3A cells expressing untagged STAT1 in combination with WT-GFP or QM-GFP (10 μl each) incubated for 0, 15 and 30 min with 2 U of the STAT-specific Tc45 phosphatase (n = 3). Tyrosine dephosphorylation was followed by immunoblotting including a quantitative analysis of the phospho-tyrosine signals divided by total amount of STAT1 signal. Scale bar in A marks a distance of 10 µm

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Plasmid Preparation, Staining, Fluorescence, Expressing, Two Tailed Test, Concentration Assay, Inhibition, Western Blot, Recombinant, In Vitro, Incubation, De-Phosphorylation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Unaltered DNA binding, reporter activation and target gene induction by WT STAT1 in the presence of a dimerization-deficient mutant. A Electrophoretic mobility shift assay demonstrated unchanged binding of the recombinant WT protein from reconstituted U3A cell extracts to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, whereas the co-expressed unphosphorylated STAT1 variants and empty vector (pEGFPN1) showed no DNA-binding indicated by the absence of a GFP-tagged STAT1 band in lanes 3 to 5. The asterisk at the margin of the gelshift indicates an unspecific band. B EMSA result showing the unaltered capacity of endogenous STAT1 from HeLa cell extracts to form tetramers on the radioactively-labelled DNA probe 2xGAS, containing two GAS sites in tandem orientation, in the presence of co-expressed QM-GFP. HeLa cells were either untransfected (UT) or transfected with the indicated expression plasmids, and on the next day the cells were treated for 45 min with IFNγ or left untreated. Extracts were incubated with 2xGAS DNA probed for 15 min before subsequently being challenged by a 750-fold molar excess of unlabelled M67 DNA incubated for 10 min at RT in a competition assay (comp). For super-shift assays, 20 ng of STAT1- (αS1) or STAT3-specific (αS3) antibody were added to the reaction for 40 min at RT. C , D EMSA result showing the unaltered cytoplasmic and nuclear fractions of endogenous P-STAT1 from HeLa cell lysates bound to a [ 33 P]-radioactively labelled M67 probe containing a single GAS sequence, as the additional presence of QM-GFP did not hamper nuclear import of the endogenous STAT1 but its nuclear retention. E HeLa cells expressing WT-GFP, empty vector (pEGFPN1) or the quadruple mutant QM-GFP were left untreated (−) or stimulated for 6 h with 50 ng/ml of IFNγ (+). In whole extracts from these cells, luciferase luminescence of a reporter construct with a triple GAS site (3xLy6E) and the enzymatic activity of the co-expressed β-galactosidase were measured and represented graphically. F U3A cells were transfected with a STAT1-responsive luciferase reporter construct, a β-galactosidase expression vector and a combination of equal amounts of WT-GFP and the indicated STAT1 variants. These cells were either unstimulated (−) or stimulated with 50 ng/ml of IFNγ (+) and subsequently luciferase activity, normalized to the β-galactosidase expression, was measured in whole cell extracts and represented graphically. The experiment was repeated in six independent transfections at least three times. G – J U3A cells were untransfected (UT) or transfected with a plasmid coding for WT-GFP alone or a combination of WT-GFP and the indicated U-STAT1 variants or empty GFP vector (pEGFPN1). These cells where either untreated or stimulated with 50 ng/ml of IFNγ for 6 h. after which RNA was isolated and converted to cDNA. Histograms show the results from qPCR experiments for the following STAT1 target genes: G GBP1 , H MIG , I CXCL10 and J IRF1 . Gene induction was normalized to the expression of the house-keeping gene GAPDH . Histograms show means and standard deviations wherein the significant differences between the IFNγ-stimulated variant samples and cells expressing a single transfection of WT protein are marked by bars and asterisks. The experiment was repeated three times

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Binding Assay, Activation Assay, Mutagenesis, Electrophoretic Mobility Shift Assay, Recombinant, Sequencing, Plasmid Preparation, Transfection, Expressing, Incubation, Competitive Binding Assay, Luciferase, Construct, Activity Assay, Isolation, Variant Assay

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Deletion of the amino-terminus in mutant U-STAT1 restores nuclear accumulation of co-expressed, endogenous P-STAT1. A , B HeLa cells were transfected with an expression plasmid coding for R602L/Y701F-GFP, QM-GFP, ΔN/R602L/Y701F-GFP and QM/L407A/L409A-GFP and stimulated for 0 min or 45 min with 50 ng/ml of recombinant IFNγ, followed by staining with an anti-phospho-tyrosine antibody. Fluorescence micrographs show restored nuclear accumulation of P-STAT1 in the presence of amino-terminal deletion mutant ΔN/R602L/Y701F-GFP and the dsNLS mutant QM/L407A/L409A-GFP. Histograms show the quantification of nuclear P-STAT1 from HeLa cells co-expressing the indicated STAT1 variants in comparison with untransfected HeLa cells from three independent experiments (n = 3, means ± standard deviations from n = 20 cells, * p ≤ 0.05). C Fluorescence micrographs displaying the absence of nuclear endogenous P-STAT1 staining in the presence of mutant U-STAT1 constructs expressed in HeLa cells stimulated for 0 min and 45 min with recombinant IFNα (50 ng/ml). Note the rescue effect mediated by the deletion of the amino-terminus in the construct ΔN/R602L/Y701F-GFP under stimulation with type I IFN. Scale bars in A , C mark a distance of 10 µm. D Cellular fractionation experiments using cytoplasmic and nuclear lysates from HeLa cells expressing the indicated STAT1 variants demonstrate the presence of endogenous phospho-STAT1 in both compartments in IFNγ-pretreated cells. Representative immunoblots demonstrate unchanged tyrosine phosphorylation of endogenous STAT3 in whole cell extracts from HeLa cells expressing U-STAT1, after stimulation with IFNγ (lower panels). E , F Quantification from immunoblots of whole extracts from HeLa cells expressing the indicated mutants, including the quadruple mutant (QM), before and after treatment for 45 min with 50 ng/ml of recombinant IFNγ from three independent transfection experiments for the expression of phospho-STAT1 ( E ) and the expression of phospho-STAT3 ( F )

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Mutagenesis, Transfection, Expressing, Plasmid Preparation, Recombinant, Staining, Fluorescence, Comparison, Construct, Cell Fractionation, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form

doi: 10.1186/s12964-022-00841-3

Figure Lengend Snippet: Proposed model of U-STAT1-dependent import modulation of P-STAT1. The left panel describes a condition of low-level cellular U-STAT1, wherein fully functional import complexes of P-STAT1 are formed and translocated to the nucleus in cytokine-stimulated cells. The right panel depicts a high concentration of U-STAT1, wherein U-STAT1 interacts through its amino-terminus and dsNLS with both components of the importin dimer. The formation for a full-fledged import complex is hindered by the missing tyrosine phosphorylation residue of U-STAT1

Article Snippet: The membranes were incubated with either a rabbit monoclonal phospho-Tyr701-specific STAT1 antibody (Cell Signaling Technology, 58D6) or monoclonal pan-STAT1 antibody (Cell Signaling Technology, D1K9Y) overnight.

Techniques: Functional Assay, Concentration Assay, Phospho-proteomics, Residue

Journal: Journal of Neuroinflammation

Article Title: A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation

doi: 10.1186/s12974-017-0906-6

Figure Lengend Snippet: Identification of Kv1.3-regulated signaling mechanisms in LPS-induced microglial activation. a , b Canonical pathway analysis of 120 Kv1.3-dependent proteins identified in our proteomic dataset revealed highly represented signaling pathways, two of which are shown here (see Additional file : Table S5 for others). Members of this list of 120 proteins are marked with ( red circle ). Transcription factors are also highlighted ( transparent red circle ). Arrows indicate directionality of the interaction (upstream vs. downstream). a GABPA, a Kv1.3-dependent transcription factor that was downregulated by LPS, was placed upstream of several Kv1.3-regulated proteins. b MHCI proteins of relevance to our results (TAP1, Tapasin, and EHD1 proteins) were represented in a signaling network that suggested that STAT1 and IRF1 may serve as upstream regulators of these proteins. c Comparison of normalized protein expression of transcription factors identified in our proteomic dataset across treatment groups. d Quantitative RT-PCR data comparing IRF1, IRF7, and NFKB1 mRNA expression across treatment groups (paired t tests were used for these comparisons; six replicates/group). e Phospho-flow cytometric studies of serine (S727) and tyrosine (Y701) STAT1 phosphorylation in BV2 microglia ( n = 5/treatment group) at 30-min and 3-h time points. f Immunofluorescence microscopy showing partial co-localization between Kv1.3 ( green , detected by ShK-F6CA labeling) and CD14 ( red ) in LPS-activated BV2 microglia (* p < 0.05, ** p < 0.01, *** p < 0.005)

Article Snippet: Phospho-flow cytometry was performed to detect pS727 and pY701 STAT1 phosphorylation (pY701-Alexa488 ab: Cell Signaling, pS727-Alexa647: BD Biosciences) [ ].

Techniques: Activation Assay, Protein-Protein interactions, Comparison, Expressing, Quantitative RT-PCR, Phospho-proteomics, Immunofluorescence, Microscopy, Labeling

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: si-RNA sequences.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Sequencing, Negative Control

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: Sequences of primers used for reverse transcription-quantitative PCR.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 binds to the BST2 promoter to regulate BST2 expression in oral squamous cell carcinoma. (A) JASPAR and UCSC databases were used to predict the binding sites between STAT1 and the BST2 promoter. (B) mRNA expression levels of STAT1 and BST2 in SCC-15 cell lines were assessed using reverse transcription-quantitative PCR after regulation of STAT1 or BST2. (C) Relative luciferase activity was assessed using the dual-luciferase reporter assay in SCC-15 cells co-transfected with oe-STAT1 or oe-NC and BST2-WT or BST2-MUT. ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; oe, overexpression; WT, wildtype; MUT, mutant; NS, not significant; UCSC, University of California Santa Cruz Genome Browser.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Reporter Assay, Transfection, Over Expression, Mutagenesis

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 is highly expressed in HNSC and OSCC cell lines. (A) The mRNA expression levels of STAT1 from the Gene Expression Profiling Interactive Analysis database in different tumors. (B) Differential expression of STAT1 between normal and tumor tissues (tumor tissues, n=519; normal tissues, n=44). (C) Kyoto Encyclopedia of Genes and Genomes pathway analysis performed using the linkedomics database. (D) The protein and mRNA expression levels of STAT1 in different OSCC cell lines. * P<0.05 and *** P<0.001. HNSC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; TPM, transcripts per million; FDR, false discovery rate; NS, not significant; T, tumor; N, normal.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 positively regulates BST2/AKT/ERK1/2 and biological behavior of OSCC. (A) Western blotting analysis of the protein expression levels of STAT1, BST2, AKT and ERK1/2 and the extent of AKT and ERK1/2 phosphorylation after using si-RNA or oe plasmid to regulate STAT1 or BST2 in SCC-15 cells. (B) Cell scratch test assay (magnification, ×50), and (C) Transwell assay (magnification, ×100) were used to assess the migration and invasion ability of SCC-15 cells after STAT1 downregulation. (D) Clone formation and (E) cell proliferation assays were performed to assess changes in the viability and proliferation of SCC-15 cells after STAT1 downregulation. * P<0.05, ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; si, small interfering; oe, overexpression; NC, negative control; p, phosphorylated; NS, not significant.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Western Blot, Expressing, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Migration, Over Expression, Negative Control

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 and BST2 serve a synergistic role in the biological behavior of oral squamous cell carcinoma. (A) Western blotting analysis of the protein expression levels of STAT1, BST2, AKT, ERK1/2 and the extent of AKT and ERK1/2 phosphorylation after using si-RNA or oe plasmid to regulate STAT1 or BST2 in SCC-15 cells. (B) Cell scratch test assay (magnification, ×50), (C) Transwell assay (magnification, ×100) and (D) clone formation assay were used to assess the migration, invasion and proliferation of SCC-15 cells after using si-RNA or oe plasmid to regulate STAT1 or BST2. * P<0.05, ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; si, small interfering; oe, overexpression; NC, negative control; p, phosphorylated; NS, not significant.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Western Blot, Expressing, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Migration, Over Expression, Negative Control

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: (A) Photograph of tumors excised from mice, growth curve of subcutaneous tumor volume, and the weight of mice. (B) The protein expression levels of STAT1, BST2, AKT and ERK1/2 and the extent of STAT1, AKT and ERK1/2 phosphorylation in vivo after downregulation of STAT1 (n=3 selected from each group) and mRNA expression levels of STAT1 and BST2. Representative images of (C) hematoxylin and eosin and (D) immunohistochemical staining for STAT1, p-STAT1 and BST2 of oral squamous cell carcinoma tumor samples (magnification, ×200). * P<0.05, ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; si, small interfering; oe, overexpression; NC, negative control; p, phosphorylated; NS, not significant.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Expressing, In Vivo, Immunohistochemical staining, Staining, Over Expression, Negative Control

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay