Journal: Journal of Biological Chemistry

Article Title: Inhibition of p38 Mitogen-activated Protein Kinase Impairs Influenza Virus-induced Primary and Secondary Host Gene Responses and Protects Mice from Lethal H5N1 Infection

doi: 10.1074/jbc.m113.469239

Figure Lengend Snippet: FIGURE 4. p38 MAPK inhibition affects H5N1-induced IFN expression. A, impact of p38 MAPK inhibition on the IFN promoter activity. Vero cells were transfected with the IFN promoter for 24 h. Cells were preincubated with 20 M SB 202190 or left untreated and subsequently stimulated with 500 ng of total RNA isolated from infected A549 cells (8 h, 5 m.o.i.). Total RNA from uninfected A549 cells was used as control. 5 h p.s. promoter activity was measured by a luciferase assay and the results are depicted as mean n-fold ( S.D.) of three independent experiments normalized to controls. B, Western blot analysis of total lysates of HUVEC treated with UV-inactivated, filtered conditioned media from mock-infected control cells (lanes 5 and 7) and KAN-1-infected cells (5 m.o.i., 5 h) (lanes 6 and 8). Donor cells were pretreated with DMSO (lanes 1 and 3) or SB 202190 (20 M, lanes 2 and 4). STAT1 Tyr701 phosphorylation was detected 15 min aftertreatmentwithconditionedmedium(upperpanel).EqualloadingwasverifiedbythedetectionoftotalERK2(lowerpanel).Blotsarerepresentativeofthree independent experiments.

Article Snippet: The expression plasmid pRC/CMV STAT1 Y701F was obtained from Addgene, provided by J. Darnell (Laboratory of Molecular Cell Biology, Rockefeller University, New York) and previously described (24).

Techniques: Inhibition, Expressing, Activity Assay, Transfection, Isolation, Infection, Control, Luciferase, Western Blot, Phospho-proteomics

Journal: mSphere

Article Title: Interactions of the Nipah Virus P, V, and W Proteins across the STAT Family of Transcription Factors

doi: 10.1128/mSphere.00449-20

Figure Lengend Snippet: Interaction with STAT4 is conserved across NiV P, V, and W, whereas STAT5 specifically interacts with NiV V. A coimmunoprecipitation (co-IP) assay was performed on HEK293T cells transfected with plasmids encoding the indicated HA-tagged NiV P, V, W, and P N terminus (PNT) protein (2 μg) and FLAG-tagged STAT1 (A), STAT2 (B), STAT3 (C), STAT4 (D), STAT5a and -5b (E), and STAT6 (F) (2 μg). pCAGGS denotes the empty vector control. Co-IP was performed using anti-HA beads. Western blots for NiV and STAT protein expression in whole-cell lysates (WCL) and anti-HA bead elutions (IP: HA) were performed using anti-HA and anti-FLAG antibodies as indicated. Anti-GAPDH blots served as a loading control for the WCL. The immunoblots (IB) are representative of at least three independent experiments.

Article Snippet: Mouse anti-STAT1 and anti-phospho(Y701F)-STAT1 were purchased from BD Transduction Laboratories.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: mSphere

Article Title: Interactions of the Nipah Virus P, V, and W Proteins across the STAT Family of Transcription Factors

doi: 10.1128/mSphere.00449-20

Figure Lengend Snippet: NiV V C-terminus interaction with STAT5 modulates its activity. (A) Co-IP was performed on 293T cells transfected with plasmids encoding FLAG-tagged STAT5b and empty vector control (pCAGGS) or GST alone or fused to the unique C terminus of NiV V or NiV W (GST-VCT and GST-WCT) using glutathione magnetic beads. Western blots were performed for FLAG and GST protein expression in whole-cell lysates (WCL) and bead elutions (IP: GST); the immunoblots (IB) are representative of two independent experiments. (B) A co-IP assay was performed using anti-HA beads on 293T cell lysate transfected with HA-tagged NiV V, FLAG-tagged STAT5a or -5b, and increasing concentrations of MDA5, as indicated. Western blots were performed for anti-HA and anti-FLAG. (C) A co-IP assay was performed as in panel B, on 293T lysates transfected with HA-tagged NiV V, FLAG-tagged STAT5a or -5b, and increasing concentrations of GFP-tagged STAT1. (D) 293T cells were transfected with increasing concentrations of FLAG-tagged STAT5b in 10-fold steps (0 to 20 ng), constitutively expressed Renilla luciferase reporter, STAT5 response element (STAT5RE)-firefly luciferase reporter plasmid, and HA-tagged NiV V, as indicated. Cells were treated with IFN-β and ruxolitinib, as indicated. The firefly luciferase signal was normalized to the Renilla luciferase signal, and the fold increase over mock-treated samples was determined. Error bars represent standard errors of four transfections performed in parallel. The experiment was performed three times. Statistical significance was determined by using a two-tailed t test (*; P < 0.05; ***; P < 0.001). E, empty vector control; V, transfection with HA-NiV V plasmid. (E) Western blot of the panel D luciferase assay samples treated with IFN-β and ruxolitinib, as indicated. Expression of HA-NiV V, Flag-STAT5b, and the phosphorylation status of STAT5b was assessed by Western blotting. IB, immunoblot.

Article Snippet: Mouse anti-STAT1 and anti-phospho(Y701F)-STAT1 were purchased from BD Transduction Laboratories.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Magnetic Beads, Western Blot, Expressing, Luciferase, Two Tailed Test

Journal: mSphere

Article Title: Interactions of the Nipah Virus P, V, and W Proteins across the STAT Family of Transcription Factors

doi: 10.1128/mSphere.00449-20

Figure Lengend Snippet: N-terminal residues 114 to 140 are necessary for efficient NiV P, V, and W inhibition of STAT1 and STAT4 activity. (A and B) HEK293T cells were transfected with an ISG54-promoter firefly luciferase reporter (A) or a STAT4-response element (STAT4RE) firefly luciferase reporter (B), constitutively expressed Renilla reporter and the indicated NiV P, V, or W wild-type (WT), the 114-140 deletion mutant (Δ), or a G121E point mutant (121) expression plasmid. E, empty vector control. Transfected cells were treated with IFN-β or IFN-γ, as indicated. At 24 h posttreatment, the firefly luciferase activity was normalized to the Renilla luciferase activity, and the fold increase over mock treatment was determined. Error bars represent standard errors of four transfections performed in parallel. The experiment was performed three times. Statistical significance was determined by using a two-tailed t test for the indicated samples (****; P < 0.0001). NiV protein expression was confirmed by Western blotting with anti-HA and anti-GAPDH. IB, immunoblot.

Article Snippet: Mouse anti-STAT1 and anti-phospho(Y701F)-STAT1 were purchased from BD Transduction Laboratories.

Techniques: Inhibition, Activity Assay, Transfection, Luciferase, Mutagenesis, Expressing, Plasmid Preparation, Two Tailed Test, Western Blot

Journal: mSphere

Article Title: Interactions of the Nipah Virus P, V, and W Proteins across the STAT Family of Transcription Factors

doi: 10.1128/mSphere.00449-20

Figure Lengend Snippet: GST‐114-140 interacts with the SH2 domain of STAT1. (A) Schematic of STAT1β domains and the FLAG‐tagged STAT1/STAT3 chimeras. STAT1 comprises the N‐terminal domain (ND), coiled‐coil domain (CCD), DNA‐binding domain (DBD), linker domain (LD), Src‐homology 2 domain (SH2), and transactivation domain (TD). Larger domains are divided into N‐ and C‐terminal halves (NT and CT, respectively). (B) A co-IP was performed on HEK293T cells that were transfected with GFP, STAT1α, STAT1β, or STAT3 or the indicated STAT1/STAT3 chimeras and a plasmid encoding GST‐114-140. Co-IP was performed using glutathione magnetic beads. Western blotting was performed for FLAG and GST in whole-cell lysates (WCL) and bead elutions (IP: GST) as previously described. The immunoblots (IB) are representative of two independent experiments.

Article Snippet: Mouse anti-STAT1 and anti-phospho(Y701F)-STAT1 were purchased from BD Transduction Laboratories.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Magnetic Beads, Western Blot

Journal: mSphere

Article Title: Interactions of the Nipah Virus P, V, and W Proteins across the STAT Family of Transcription Factors

doi: 10.1128/mSphere.00449-20

Figure Lengend Snippet: NiV P 111-140 peptide construct interacts with STAT1 and STAT4 and impairs STAT1 phosphorylation. (A) A co-IP was performed on HEK293T cells transfected with either an empty control plasmid (pCAGGS) or GST fusions to NiV P amino acid residues 111 to 140 or 114 to 140, and FLAG-tagged constructs of STAT1, STAT3, STAT4, or STAT5b, as indicated. Co-IP assays were performed with glutathione magnetic beads; Western blots were performed for FLAG and GST in whole-cell lysates (WCL) and elutions (IP: GST). The immunoblots (IB) are representative of two independent experiments. (B) HEK293T cells were transfected with either empty vector (pCAGGS) or GST-111-140 or GST-111-140 peptide with amino acid residues 114 to 116 replaced with alanines (111-AAA-140) or amino acid residues 114 to 140, derived from the N terminus of NiV P, and FLAG-tagged STAT1 or STAT4. Co-IPs were performed using glutathione magnetic beads; bead elutions (IP: GST) and lysates (WCL) were probed for FLAG and GST. The immunoblots (IB) are representative of two independent experiments. (C and D) HEK293T cells were transfected with increasing concentrations (2-fold dilutions, 32 to 250 ng) of plasmids expressing GST-111-140, GST-111-AAA-140, GST-114-140, or GST alone, as indicated. Cells were treated with IFN-β and ruxolitinib (rux) as indicated, for 30 min (C) or 24 h (D). Western blots were performed for GST, STAT1, and phosphorylated STAT1 (pSTAT1). The percent pSTAT1 was determined relative to the GST only control, which was set at 100%. Statistical significance, relative to the GST control, was determined by ANOVA with Dunnett’s multiple-comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). The assay was performed in triplicate; error bars represent the standard errors for each triplicate. One representative Western blot is shown.

Article Snippet: Mouse anti-STAT1 and anti-phospho(Y701F)-STAT1 were purchased from BD Transduction Laboratories.

Techniques: Construct, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Magnetic Beads, Western Blot, Derivative Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: Dual Phosphorylation of STAT1 at Y701/S727 by TNFα Drives AIM2-Mediated PANoptosis of Renal Tubular Epithelial Cells and Fibrotic Progression in Renal Allografts

doi: 10.7150/ijbs.123441

Figure Lengend Snippet: Phosphorylated STAT1 modulates AIM2 expression and RTEC PANoptosis. A ) Venn diagram of differentially expressed genes (DEGs) among the GSE9493 and GSE76882 datasets, murine renal allograft RNA-seq (Syn vs Allo), and HK-2 cell RNA-seq (control vs TNF-α). B ) Venn diagram of transcription factors whose putative binding sites within the AIM2 promoter were predicted by hTFtarget, KnockTF, JASPAR, and GTRD. C ) Overlap between DEGs and predicted transcription factors. D-E ) Western blot analysis of the protein levels of STAT1 and p-STAT1 in renal tissue from normal, CAD, Syn, and Allo groups. F ) Representative IHC images of p-STAT1 expression in renal tissue from normal, CAD, Syn, and Allo groups. G-H ) Western blot and quantitative analysis of the protein levels of STAT1, p-STAT1 (Tyr701), and p-STAT1 (Ser727) in HK-2 cells treated with TNF-α (100 ng/mL) for 0, 0.25, 0.5, 1, 3, or 6 h. I-J ) Western blot analysis of the protein levels of ( I ) STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), AIM2, and Fibronectin; ( J ) full-length/cleaved GSDMD, full-length/cleaved CASP1, full-length/cleaved CASP3, and p-MLKL in HK-2 cells treated with TNF-α supplemented with or without Fludarabine (10 μM). K-L ) Western blot analysis of the protein levels of ( K ) STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), AIM2, and Fibronectin; ( L ) full-length/cleaved GSDMD, full-length/cleaved CASP1, full-length/cleaved CASP3, and p-MLKL from the control groups (NC) and STAT1 knockdown groups (Ri-STAT1) treated with TNF-α. M ) Representative IF images of STAT1 and p-STAT1 expression in HK-2 cells from the control and TNF-α-treated groups. N ) Western blot analysis of the protein levels of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in cytoplasmic and nuclear extracts of HK-2 cells treated with TNF-α. O ) Western blot analysis of the protein levels of STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727) from the control groups (WT) and STAT1 phospho-site mutant (STAT1-Y701F, and STAT1-S727A) groups treated with TNF-α. P ) Western blot analysis of the protein levels of STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), and AIM2 from the control groups (WT) and STAT1 phospho-site mutant (STAT1-Y701F, and/or STAT1-S727A) treated with TNF-α.

Article Snippet: Additionally, STAT1 phospho-site mutant plasmids (STAT1-Y701F [tyrosine→phenylalanine], STAT1-S727A [serine→alanine]) and the wild-type STAT1 plasmid were obtained from Genechem (Shanghai, China).

Techniques: Expressing, RNA Sequencing, Control, Binding Assay, Western Blot, Knockdown, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Dual Phosphorylation of STAT1 at Y701/S727 by TNFα Drives AIM2-Mediated PANoptosis of Renal Tubular Epithelial Cells and Fibrotic Progression in Renal Allografts

doi: 10.7150/ijbs.123441

Figure Lengend Snippet: TNF-α induces STAT1 phosphorylation at Tyr701 to directly bind the AIM2 promoter and enhance transcription, and phosphorylation at Ser727 via the p38/MAPK pathway further augments AIM2 transcription. A ) STAT1 DNA-binding motif obtained from JASPAR. B ) Predicted STAT1-binding sites within the AIM2 promoter (JASPAR). C-E ) HK-2 cells were treated with TNF-α (100 ng/mL) or vehicle for 24 h, then subjected to ChIP using anti-STAT1 antibody; IgG served as the negative control. ( C , D ) qPCR quantification of STAT1 enrichment at site 1 and site 2. ( E ) Agarose-gel verification of site-1 amplification. F ) Relative luciferase activity of cells transfected with pGL3-AIM2-WT reporter plasmid and mutant plasmids (pGL3-AIM2-MUT1 or pGL3-AIM2-MUT2) after treatment ± TNF-α. G-K ) Western blot analysis of the protein levels of JNK, p-JNK, ERK, p-ERK, p38MAPK, p-p38MAPK, p65, p-p65, AKT, and p-AKT in HK-2 cells treated with TNF-α (100 ng/mL) for 0, 0.25, 0.5, 1, 3, and 6 h. L ) Western blot analysis of AIM2 in HK-2 cells treated with TNF-α supplemented with or without (MK-2206 [AKT inhibitor], QNZ [NF-κB inhibitor], SB203580 [p38MAPK inhibitor], SP600125 [JNK inhibitor], or U0126 [MEK/ERK inhibitor]) for 24 h. M ) Western blot and quantitative analysis of Fibronectin, STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), and AIM2 in HK-2 cells treated with TNF-α supplemented with or without SB203580. N ) Western blot analysis of Fibronectin, STAT1, p-STAT1 (Tyr701), p-STAT1 (Ser727), and AIM2 in HK-2 cells treated with TNF-α supplemented with or without R7050 (TNF-α Antagonist).

Article Snippet: Additionally, STAT1 phospho-site mutant plasmids (STAT1-Y701F [tyrosine→phenylalanine], STAT1-S727A [serine→alanine]) and the wild-type STAT1 plasmid were obtained from Genechem (Shanghai, China).

Techniques: Phospho-proteomics, Binding Assay, Negative Control, Agarose Gel Electrophoresis, Amplification, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Dual Phosphorylation of STAT1 at Y701/S727 by TNFα Drives AIM2-Mediated PANoptosis of Renal Tubular Epithelial Cells and Fibrotic Progression in Renal Allografts

doi: 10.7150/ijbs.123441

Figure Lengend Snippet: Schematic description of the molecular mechanism. TNF-α drives tubular epithelial injury via the direct induction of STAT1 Y701 phosphorylation and p38 MAPK-mediated STAT1 phosphorylation at S727, cooperatively upregulating AIM2 to execute PANoptosis. PANoptotic cells secrete IL-6 and TGF-β, inducing EMT in adjacent tubular cells, thereby collectively promoting renal allograft interstitial fibrosis.

Article Snippet: Additionally, STAT1 phospho-site mutant plasmids (STAT1-Y701F [tyrosine→phenylalanine], STAT1-S727A [serine→alanine]) and the wild-type STAT1 plasmid were obtained from Genechem (Shanghai, China).

Techniques: Phospho-proteomics