Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV results in activated phosphorylation of STAT3 on Tyr705 and diaphragm contractile dysfunction. Diaphragms from mechanically ventilated (MV) rats and unfed controls were analyzed. A ) Ex vivo force–frequency relationship from mechanically ventilated ( n =13) rats and unfed controls ( n =7). B ) Western blots for total STAT3 and phospho-STAT3 Y705 from a representative set of mechanically ventilated rats and unfed controls (1, 3, 6, and 9 h, n =5–6 rats/group; 18 h, n =6–8). C ) Messenger RNA levels of the STAT3 downstream target genes SOCS3 and Myf5 from mechanically ventilated ( n =9) rats and unfed controls ( n =10). Mechanical ventilation period of 18 h. Results are means ± sem . * P < 0.05; Student's t test.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Ex Vivo, Western Blot

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV increases Ser705 phosphorylation and mitochondrial accumulation of phospho-STAT3 (Ser727 and/or Tyr705/Ser727) within diaphragm muscle. A ) Diaphragms from unfed control rats ( n =8), MV-R548 rats ( n =9), or MV-Veh rats ( n =9) were analyzed by Western blot of total muscle lysate (representative animals depicted in blot) and quantitation. Correlation between increases in phospho-STAT3 S727 and phospho-STAT3 Y705 were determined. B ) Purified mitochondrial fractions (mito) and total lysates (total) were subjected to Western blot analysis for pSTAT3 S727 , pSTAT3 Y705 , total STAT3, GRIM-19, VDAC (mitochondrial marker), LDHA (cytoplasmic marker) and PCNA (nuclear marker). Unfed (U) controls, n = 10; MV-Veh (MV), n = 6; MV-R548, n = 7. Results are means ± sem . Mechanical ventilation period of 18 h. P values calculated by 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Western Blot, Quantitation Assay, Purification, Marker

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: Proposed model for role of JAK signaling in VIDD. JAK signaling is activated by CMV and functions as a critical triggering mechanism upstream of STAT3 phosphorylation (Tyr705 and Ser705), mitochondrial dysfunction, ROS production, atrophy, and muscle weakness. Mechanical ventilation results in the mitochondrial accumulation of phospho-STAT3 (singly phosphorylated on Ser727 or doubly phosphorylated at both Ser727 and Tyr705). Import into mitochondria is facilitated through interaction of phospho-STAT3 with GRIM-19, a component of complex I of the ETC, and may directly affect mitochondrial function and ROS generation. Induction of various myogenic transcription factors and muscle-specific E3 ubiquitin ligases (MURF-1 and atrogin-1) and activation of calpain, caspase 9, and caspase 3 can contribute to muscle atrophy and proteolytic cleavage of myofilament proteins. Mitochondrial dysfunction may also lead to ROS-mediated modification of myofilament proteins in a manner that negatively affects contractile function.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Activation Assay, Modification

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 regulates STAT3 activation. ( A ) NIH-3T3 cells were transfected with either the mock or Ssu72 overexpression vector and stimulated with IL-6 (20 ng/ml) for 1 h. Cells were used to detect the p-STAT3 Tyr705 and p-STAT3 Ser727 levels. ( B ) NIH-3T3 cells were stained with DAPI (blue) and antibodies against tubulin (green) and p-STAT3 Tyr705 (red). Confocal scanning microscopy was performed to detect the p-STAT3 Tyr705 and p-STAT3 Ser727 levels in the transfected cells. ( C ) NIH-3T3 cells were transfected with the expression vector and stimulated with IL-6 (20 ng/ml) for 1 h. ( D ) Cells were transfected with STAT3 overexpression vectors and either the mock or Ssu72 overexpression vector. The expression levels of the Il17a mRNA were measured using real-time PCR. ( E ) NIH-3T3 cells were transfected with the Il17a promoter construct and either mock or Ssu72 expression vectors. Luciferase activity was then detected. ( F ) Lysates from the transfected NIH-3T3 cells were immunoprecipitated with the anti-FLAG antibody and immunoblotted with anti-p-STAT3 Tyr705, anti-p-STAT3, and anti-Ssu72 antibodies. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, ***P < 0.01).

Article Snippet: Membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with antibodies against p-STAT3 Y705, p-STAT3 S727, total STAT3 (Cell Signaling Technologies), Ssu72, β-actin (Santa Cruz Biotechnology), or FLAG (Sigma-Aldrich) overnight at 4 °C.

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Staining, Microscopy, Expressing, Real-time Polymerase Chain Reaction, Construct, Luciferase, Activity Assay, Immunoprecipitation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 controls inflammatory responses in vitro . ( A ) NIH-3T3 cells were transfected with a control siRNA or Ssu72 siRNA and stimulated with IL-6 (20 ng/ml) for 0.5 h. Cells were used to examine the p-STAT3 Tyr705 and p-STAT3 Ser727 levels. ( B ) Expression levels of the Ssu72 mRNA in cells transfected with the siRNAs were measured by real-time PCR. ( C ) NIH-3T3 cells were transfected with the Il17a promoter construct and either the siRNA control or siRNA Ssu72 to detect luciferase activity. ( D ) NIH-3T3 cells were transfected with siRNAs and stimulated with IL-6 (20 ng/ml) for 0.5 h. Real-time PCR was performed to measure the expression levels of the IL-1β , IL-17A , IL-21 , TBK1 , and IKBKE mRNAs. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01).

Article Snippet: Membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with antibodies against p-STAT3 Y705, p-STAT3 S727, total STAT3 (Cell Signaling Technologies), Ssu72, β-actin (Santa Cruz Biotechnology), or FLAG (Sigma-Aldrich) overnight at 4 °C.

Techniques: In Vitro, Transfection, Expressing, Real-time Polymerase Chain Reaction, Construct, Luciferase, Activity Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 ameliorates the development of CIA. ( A ) The Ssu72 overexpression or mock vector was administered systemically to mice with CIA once per week. The clinical scores and incidence of CIA were measured in these mice (***P < 0.01, n = 10). ( B ) Total IgG levels were measured in each group (***P < 0.01, n = 10). ( C ) The levels of p-STAT3 Tyr705 and p-STAT3 Ser727 in splenocytes from mice with CIA (mock or Ssu72-overexpressing) that had been stimulated with IL-6 (20 ng/ml) for 1 h were examined by western blot analysis. ( D ) Joint tissues from mice with CIA were stained with hematoxylin and eosin (original magnification, 40× or 200×, n = 6) and Safranin O (original magnification, 40×, n = 6). ( E ) Immunohistochemical staining was used to detect IL-6, IL-21, IL-17, IL-1β, TNF-α and RANKL in the synovium of mice with CIA (mock or Ssu72-overexpressing; (original magnification, 200× or 400×, n = 6). The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01).

Article Snippet: Membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with antibodies against p-STAT3 Y705, p-STAT3 S727, total STAT3 (Cell Signaling Technologies), Ssu72, β-actin (Santa Cruz Biotechnology), or FLAG (Sigma-Aldrich) overnight at 4 °C.

Techniques: Over Expression, Plasmid Preparation, Western Blot, Staining, Immunohistochemical staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Ssu72 attenuates autoimmune arthritis via targeting of STAT3 signaling and Th17 activation

doi: 10.1038/s41598-017-05421-x

Figure Lengend Snippet: Ssu72 reduces STAT3 activation and Th17 cell differentiation. ( A ) Splenocytes from mice with CIA were stimulated with IL-6 (20 ng/ml) for 1 h and then treated with either GST or GST-Ssu72. ( B ) Splenocytes from mice with CIA were cultured under Th17 conditions for 72 h and then the number of CD4 + IL-17 + cells was quantified. Statistical analyses were conducted using one-way ANOVA with Bonferroni’s post hoc test. ( C ) IL-17 and TNF-α expression in culture supernatants was measured using ELISA. The data represent the mean ± SD from three independent experiments. Statistical analyses were conducted using the nonparametric Mann-Whitney U -test (*P < 0.05, **P < 0.03, ***P < 0.01, n = 5).

Article Snippet: Membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and incubated with antibodies against p-STAT3 Y705, p-STAT3 S727, total STAT3 (Cell Signaling Technologies), Ssu72, β-actin (Santa Cruz Biotechnology), or FLAG (Sigma-Aldrich) overnight at 4 °C.

Techniques: Activation Assay, Cell Differentiation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Precise and efficient C-to-U RNA base editing with SNAP-CDAR-S

doi: 10.1093/nar/gkad598

Figure Lengend Snippet: Benchmark with Cas13-RESCUE-S on endogenous targets and applications. ( A ) Comparison of editing yields at various sites on various endogenous targets and one disease-relevant cDNA (APOE) comparing SNAP-CDAR-S with standard guide RNAs (30 nt, 6-C-23, 2′OMe gapmer) versus Cas13 RESCUE-S with plasmid-borne optimized Cas guide RNAs. Both editing enzymes were expressed from the same single genomic locus. ( B ) Comparing both tools, SNAP-CADR-S versus Cas RESCUE-S, for the activation of β-catenin by RNA editing. Given are C-to-U editing yields (T41I) and the luminescence-based read-out of pathway activation. For further controls, see . ( C ) Editing of the regulatory phospho-site serine 727-to-glycine in STAT3, read-out of editing yield by Sanger sequencing, and amount of total STAT3 and pS727 STAT3 protein by western blot. Data in (A), (B) and (C) are shown as the mean ± s.d. of N = 3 independent experiments.

Article Snippet: 30 μg of total protein was run on a NovexTM WedgeWellTM 8 to 16%, Tris-glycine, 1.0 mm, Mini Protein Gel (ThermoFisher Scientific) with 200 V for 60 min. Blotting was performed with a Mini Trans-Blot Cell ® (BioRad) at 100 V for 60 min. For protein detection, membranes were incubated with monoclonal anti-β-actin antibody produced in mouse (Sigma, 1:5000 dil.) and either Stat3 (DRZ2G) Rabbit mAb (CellSignaling, 1:1000 dil.) or P-Stat3 (S727) (D8C2Z) Rabbit mAb (CellSignaling, 1:1000 dil.).

Techniques: Comparison, Plasmid Preparation, Activation Assay, Sequencing, Western Blot

Journal: Cell Death & Disease

Article Title: CXCR4 uses STAT3-mediated slug expression to maintain radioresistance of non-small cell lung cancer cells: emerges as a potential prognostic biomarker for lung cancer

doi: 10.1038/s41419-020-03280-5

Figure Lengend Snippet: A Western blot analysis of phopho-STAT3 (Y705 and S727) in NSCLC cells (A549, H460, and A549/GR) transfected with the vectors described in the Figure. B Western blot (WB, upper) and RT-PCR (lower) analysis (left) and IR clonogenic survival assay (right) of A549/GR cells transfected with control siRNA (siCon) or siRNA against STAT3 (siSTAT3-1 and 3-2). C , G γ-H2AX foci assay of A549/GR (C) and A549/CXCR4 (upper) and H460/CXCR4 (lower) ( G ) cells. Cells were exposed to IR (6 Gy) and fixed at the indicated time points in the Figure. Representative ICC images of γ-H2AX (left) and quantification of the results (right). * P < 0.05, ** P < 0.01, *** P < 0.005. White bar: 20 μm. D , H Western blot analysis of A549/GR ( D ) and A549/CXCR4 (left) and H460/CXCR4 (right) ( H ). Cells exposed to IR (6 Gy) at the indicated time points transfected with control siRNA (siCon) or siRNA against Stat3 (siSTAT3-1). E , F Western bot analysis of p-STAT3 (S727 and Y705) (left) and IR clonogenic survival assay (right) in A549 ( E ) and H460 ( F ) cells transfected with control siRNA (siCon) or siRNA against STAT3 (siSTAT3-1 and siSTAT3-3).

Article Snippet: Antibody such as p-STAT3 (S727) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). p-STAT3 (Y705) and Slug were obtained from Cell Signaling Biotechnology (Denvers, MA).

Techniques: Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Clonogenic Cell Survival Assay, Control

Journal: Cellular and Molecular Immunology

Article Title: A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells

doi: 10.1038/s41423-021-00798-2

Figure Lengend Snippet: The p40-EBI3-Fc protein suppressed Th17 cells via regulation of IL-12Rb1 and gp130 receptor signaling. A Splenocytes from C57BL/6 mice were cultured with 10 ng/mL IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL). After 1 h, protein was isolated from the cultured cells. p -STAT3 Tyr705, STAT3, p -STAT5, STAT5, and GAPDH were analyzed by western blotting. Representative western blots from one of three independent experiments are shown (bottom panel); the results are depicted as the mean ± SD of three independent experiments per group (right). B Splenocytes isolated from C57BL/6 mice were stimulated with IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL) for 15 min. CD4 + T cells expressing pSTAT3 Tyr705, pSTAT3 Ser727, or pSTAT5 were then analyzed by flow cytometry. C A IL-12Rβ1 and gp130 overexpression vector was transfected into the Ba/F3 cell line using a P4 Primary cell 4D-nucleofector TM X kit. The proliferation of Ba/F3 cells was determined with a [ 3 H] thymidine incorporation assay under p40-EBI3-Fc (0.01–1 μg/mL) treatment. D IL-12Rβ1- and gp130-expressing Ba/F3 cells were stimulated with p40-EBI3-Fc for 1 h. p -STAT5, STAT5, and GAPDH in the cell lysates were analyzed using western blotting. Representative western blots from one of three independent experiments are shown; the results represent the mean ± SD of three independent experiments per group (right). E , F Alterations in IL-17 production in anti-CD3-stimulated murine splenic CD4 + T cells induced by p40-EBI3-Fc treatment and transfection with IL-12Rβ1-, gp130-, or WSX1-siRNA. Th17 cell expression was determined by FACS, and IL-17 expression was evaluated by ELISA. Bars show the mean ± SD of three independent experiments per group (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The permeabilized cells were stained with anti-p-STAT3 Y705-PE, anti-p-STAT3-S727-PE, or anti-p-STAT5-PE (BD Pharmingen) and subjected to flow cytometric analysis (CytoFLEX; Beckman Coulter, Fullerton, CA, USA).

Techniques: Cell Culture, Isolation, Western Blot, Expressing, Flow Cytometry, Over Expression, Plasmid Preparation, Transfection, Thymidine Incorporation Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Blockade of glucagon receptor induces α-cell hypersecretion by hyperaminoacidemia in mice

doi: 10.1038/s41467-025-57786-7

Figure Lengend Snippet: a Venn diagrams showing transcription factors that can bind to the VGF promoter, as identified in the animalTFDB3.0, GTRD and TFBIND databases. b Amino acids increased VGF promoter luciferase activity in αTC1-6 cells incubated with 4 mM glutamine and alanine for 72 h ( n = 6). c Screening for transcription factors involved in amino acid-induced VGF promoter activity. αTC1-6 cells were transfected with indicated siRNA and incubated with amino acids for 72 h. Data were generated from 3 independent experiments. d Western blot analysis of p-STAT3-S727, p-STAT3-Y705 and total STAT3 protein levels in αTC1-6 cells incubated amino acids for 72 h. e Quantification of relevant protein levels in ( d ). Data were generated from 3 independent experiments. f Representative confocal images of p-STAT3-S727 expression in the pancreatic sections from WT or GCGR-KO mice. Scale bar indicates 50 μm. g Western blot analysis of p-STAT3-S727, STAT3, VGF and pro-glucagon protein levels in αTC1-6 cells treated with S3I-201 (STAT3 inhibitor). h Quantification of relevant protein levels in ( g ). Data were generated from 3 independent experiments. i . Representative confocal microscopy images of VGF and glucagon expression in αTC1-6 cells treated with amino acids alone or plus S3I-201 for 72 h. Scale bar, 10 μm. j VGF promoter activity in αTC1-6 cells transfected with empty vector, wild-type STAT3 or constitutively active STAT3 mutant (STAT3-S727D) for 72 h. Data were generated from 3 independent experiments. k Diagram of STAT3 binding sites at VGF promoter. l Chip-qPCR analysis of STAT3 binding activity at VGF promoter. Data were generated from 3 independent experiments. m Western blot analysis of protein levels after treatment with amino acids or amino acids plus rapamycin in αTC1−6 cells for 72 h. n Quantification of relevant protein levels in ( m ). Data were generated from 3 independent experiments. Data presented in ( b , c , e , h , j , l , n ) are mean ± SEM. Data in ( b , e , l ) were analyzed using two-tailed unpaired t-tests. Data in ( c , h , j , n ) were analyzed by one-way ANOVA with Bonferroni’s post hoc test. p -values < 0.05 are displayed. Source data are provided as a Source Data file.

Article Snippet: The remaining supernatant was immunoprecipitated with p-STAT3-S727 antibodies (CST, 9198).

Techniques: Luciferase, Activity Assay, Incubation, Transfection, Generated, Western Blot, Expressing, Confocal Microscopy, Plasmid Preparation, Mutagenesis, Binding Assay, ChIP-qPCR, Two Tailed Test

Journal: Nature Communications

Article Title: Blockade of glucagon receptor induces α-cell hypersecretion by hyperaminoacidemia in mice

doi: 10.1038/s41467-025-57786-7

Figure Lengend Snippet: a Upon glucagon receptor blockade, the organism increases its demand for glucagon. Several factors (particularly elevated amino acids), arise from the GCGR-deficient liver and act on pancreatic islets, leading to α cell hyperplasia and enhanced α cell secretion. All these changes collectively contribute to hyperglucagonemia. b Increased circulating amino acids (especially glutamine and alanine) activate the mTOR-STAT3 and ERK-CREB signaling pathways. STAT3 enhances VGF transcription, while CREB promotes both VGF and GCG expression. Consequently, glucagon granule biogenesis and glucagon secretion are significantly increased. Conversely, blocking amino acid-induced VGF expression by inhibiting mTOR or STAT3 activation reduces levels of the glucagon granule component VGF, thereby decreasing glucagon granule biogenesis and glucagon secretion.

Article Snippet: The remaining supernatant was immunoprecipitated with p-STAT3-S727 antibodies (CST, 9198).

Techniques: Protein-Protein interactions, Expressing, Blocking Assay, Activation Assay