Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 2 The expression of selected hypoxia-related markers (IDH1, IDH2, HIF1a, HIF1b, HIF2a, EGFR, PTEN, VEGFA, VEGFC and STAT3) in primary glioma cells GBMLe3, GBMLe4 and GBMDo2 (A) and in cryopreserved samples corresponding to the tumor used for particular primary glioma culture derivation (B) at mRNA level. The expression of mRNA was determined by RT-PCR. Data are expressed as fold increase ± SD of averages from two independent experi ments. Beta-2-microglobulin was used as a housekeeping gene. * p < 0.05 GBM26 vs. GBM43; # p < 0.05 GBM26 vs. GBM59

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 3 Comparison of tumor growth and drug accumulation in Foxn1-nu mice after implantation of glioma cell lines followed by TMZ treatment. Tumor size of implanted (n = 4) (A) U87MG IDH1wt, U87MG STAT3 KO with and without TMZ (0.9 mg/kg) treatment. Evaluation of accumulation of (B) TMZ and (C) its metabolites AIC inside the brain, tumor and plasma in tumor bearing mice with implanted U87MG IDH1wt, resp. U87MG STAT3 KO. The administration of drug (TMZ– 0.9 mg/kg) begins two weeks after implantation (from day 15. to day 28. daily). Organs were collected 15 min after last TMZ application. Confidence interval values of tumor size are shown as mean ± SD. The data of drug accumulation are expressed as ng per mg of tissue. Measurements were performed in two independent experiments

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Comparison, Clinical Proteomics

Journal: BMC cancer

Article Title: Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

doi: 10.1186/s12885-024-12221-w

Figure Lengend Snippet: Fig. 4 The expression of selected markers related with hypoxia (IDH1, IDH2, HIF1a, HIF1b, HIF2a, EGFR, PTEN, VEGFA, VEGFC and STAT3) in glioma U87MG and U87MG STAT3 KO glioma cell lines (A) and glioma samples collected from Foxn1-nu mice with implanted U87MG and U87MG STAT3 KO glioma cells on mRNA level (B). Tumors were collected 28 days after glioma cell implantation and processed as described in Materials and methods section

Article Snippet: Crispr/Cas STAT3 knockout cell model Glioma cells U87MG grown to 50–70% confluence were transfected with transfection mixture (gRNA vectors in Opti-MEM I, the donor DNA and Turbofectin 8.0 - the ratios of 3:1 for Turbofectin: DNA) as based on manufacturer’s protocol (STAT3 Human Gene Knockout Kit (CRISPR), CAT#: KN204922, Origene).

Techniques: Expressing

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 1. The acyloxy nitroso compounds, NCA and NCP, and the prototypical HNO donor, Angel’s salt, target STAT3. Chemical structures of (A) 1-Nitrosocyclohexyl acetate, NCA, (B) 1-nitrosocyclohexyl pivalate, NCP, and (C) Angeli’s salt (AS). doi:10.1371/journal.pone.0043313.g001

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques:

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 2. NCA and NCP inhibit LIF-induced STAT3 activation in human microvascular endothelial cells. HMEC-1 were pretreated for 1 h with vehicle (0.04% v/v DMSO), (A) 100 mM NCP, or (C) 100 mM NCP. Afterwards, cells were dosed for various times with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3 as a loading control. (B and D) Results were quantified and expressed as the ratio of phosphorylated STAT3 to total STAT3. **P,0.01 and ***P,0.001 vs. same time point control (n = 4); 2-way ANOVA and Bonferroni post-test. doi:10.1371/journal.pone.0043313.g002

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 3. NCP inhibits LIF-induced STAT3 activation in cardiac myocytes. Neonatal rat ventricular myocytes (A & B) were pretreated for 1 h with 100 mM NCP (lanes 5–8) or vehicle (0.04% v/v DMSO; lanes 1–4). Cells were then dosed with 2 ng/mL LIF for various times. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3. (A) Representative immunoblot of 4 independent experiments. (B) Compiled data analysis. Adult mouse cardiac myocytes (C & D) were pretreated for 1 h with 500 mM NCP (+) or vehicle (2). Cells were then dosed with 2 ng/mL LIF for 0, 5, or 15 min. (C) Representative immunoblot of 3 independent experiments. (D) Compiled data analysis. **P,0.01 or ***P,0.001 vs. same time point control; 2-way ANOVA and Bonferroni post-test (n = 3). doi:10.1371/journal.pone.0043313.g003

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Control

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 4. Dose response curves for inhibition of LIF-induced STAT3 activation by NCA, NCP and Angeli’s salt (AS). HMEC-1 were pretreated (A) for 1 h with various doses (02100 mM) of NCA or NCP and the same amount of vehicle (0.04% v/v DMSO) or (B) for 30 min with 02500 mM Angeli’s salt and the same amount of vehicle (50 mM NaOH). Cells were treated for 15 min with 2 ng/mL LIF. Western immunoblots of cell lysates were probed for STAT3 Y705 phosphorylation and STAT3. Results represent 2 independent experiments for both NCA and NCP and a single experiment for Angeli’s salt. doi:10.1371/journal.pone.0043313.g004

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Inhibition, Activation Assay, Western Blot, Phospho-proteomics

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 8. STAT3 possesses redox-sensitive cysteines. (A) NCA and NCP block thiolate labeling. Recombinant human STAT3 was treated with vehicle (DMSO), NCA (100 mM) or NCP (100 mM) for 1 h at room temperature and then labeled for 2 h with fluorescein-5-maleimide. Equal amounts of protein were separated by SDS-PAGE and fluorescence in the gel detected (upper panel). To ensure equal loading, Western analysis was done on each fluorescein-labeled sample. Separated proteins on nitrocellulose membranes were probed with a STAT3 antibody and imunoreactive bands quantified using the Li-COR Odyssey infrared imaging system (lower panel). Results shown are representative of 3 independent experiments. (B & C) Oxidation of STAT3 is associated with sulfenic acid formation. Purified recombinant STAT3 was immunoprecipitated and pretreated with 10 mM DTT and then treated with nothing or the oxidant o-IBZ (2.5 mM) for 1 hr at 4uC. Immunoprecipitates were processed as described under ‘‘Materials and Methods’’ to determine sulfenic acid formation (STAT3-SOH). (B) Representative blot. (C) Levels of cysteine-sulfenic acid and STAT3 were quantified by the Li-COR Odyssey Detection System. Treatment with o-IBZ resulted in a significant increase in relative sulfenic acid content. **P,0.01 vs. control, n = 3; paired Student’s t-test. doi:10.1371/journal.pone.0043313.g008

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Blocking Assay, Labeling, Recombinant, SDS Page, Fluorescence, Western Blot, Imaging, Purification, Immunoprecipitation, Control

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 9. NCP enhances STAT3 glutathionylation and dimerization. HL-1 cells were treated for 30 min with vehicle (control), 500 mM NCP, 1 mM diamide, or 500 mM NCP and 1 mM diamide together. Cell extracts were prepared. (A) Equal protein amounts of cleared extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3 and glutathionylated protein using a rabbit and mouse antibody, respectively. Immunoreactive bands were detected using Li-COR Odyssey system and secondary antibodies that produced a red (anti-rabbit) or green (anti-mouse) signal. The overlay of the red and green signals produced an orange color. Relative levels of glutathionylated STAT3 were quantified. **P,0.01, 1-way ANOVA and Dunnett’s multiple comparison test (n = 3). (B) Cells were treated as in panel A. Cell extracts were added to non-reducing Laemmli’s SDS-sample buffer and subjected to SDS-PAGE. Blots were probed for total STAT3, which showed two bands consistent with STAT3 monomers and dimers. The intensity of the higher (dimer) band relative to the lower (monomer) band for each lane was quantified. *P,0.05 and **P,0.01, 1-way ANOVA and Newman–Keuls post-test (n = 3). doi:10.1371/journal.pone.0043313.g009

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Control, SDS Page, Produced, Comparison

Journal: PloS one

Article Title: Acyloxy nitroso compounds inhibit LIF signaling in endothelial cells and cardiac myocytes: evidence that STAT3 signaling is redox-sensitive.

doi: 10.1371/journal.pone.0043313

Figure Lengend Snippet: Figure 10. Oxidative stress, NCP and diamide alter the Western blot profile of STAT3 under nonreducing conditions. (A & B) Aliquots of a cleared mouse heart homogenate were incubated for 30 min with vehicle, 500 mM NCP, 1 mM diamide, or 500 mM NCP+1 mM diamide. Samples were processed for SDS-PAGE and Western blot analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 using the Li-COR Odyssey detection system. (B) Intensity of the STAT3 band in the nonreduced sample was normalized to the intensity of the band after reduction. ***P,0.001 vs. Control, 1-way ANOVA and Newman–Keuls post-test (n = 3 mouse hearts). (C) Ratio of nonreduced to reduced STAT3 in wild type (WT) and failing (Gaq) mouse hearts. STAT3 levels in mouse myocardial tissue from WT (FVB/N) and heart failure mice (Gaq overexpressing) (n = 3) were determined via immunoblot analysis under nonreducing or reducing (3.75% b-mercaptoethanol (b-ME)) conditions. Protein loads were normalized using the direct blue 71 stained membranes (DB71). *P,0.05 (Student t-test). doi:10.1371/journal.pone.0043313.g010

Article Snippet: Fluorescein-5-maleimide was from Pierce Biotechnology (Rockford, lL USA) and recombinant human STAT3 was from OriGene Technologies (Rockville, MD).

Techniques: Western Blot, Incubation, SDS Page, Control, Staining

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: TFF1 expression alters H. pylori -induced transcriptional activation and regulation of NF-κB and STAT3 target genes. a-b Luciferase activity assay using NF-κB-Luc ( a ) and STAT3-Luc ( b ). AGS-pcDNA and AGS-TFF1 cells were co-transfected with NF-κB-Luc or STAT3-Luc and β-galactosidase plasmids. After 24 h, cells were infected with H. pylori 7.13 strain. Cells were collected 4 h after infection for NF-κB-Luc and 24 h for STAT3-Luc measurements. H. pylori 7.13 infection of AGS-pcDNA cells significantly increased the luciferase activity, which was reduced after the reconstitution of TFF1. The bar graphs represent the mean ± SEM of 3 independent experiments. c – f RT-qPCR analysis showing a decrease in mRNA expression of pro-inflammatory target genes ( IL-6,VEGF-α, IL-17 and IL-23 ) in AGS-TFF1 cells relative to AGS-pcDNA cells, following infection with H. pylori 7.13. The bars represent the mean ± SEM of three independent experiments

Article Snippet: Chromatin solutions were immunoprecipitated with NF-κB (Cat# 8242) or STAT3 (Cat# 12640) monoclonal antibodies from cell signaling (1:100 dilution) at 4 °C overnight.

Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Transfection, Infection, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: TFF1 attenuates H. pylori-induced activation of NF-κB and STAT3 in vitro. a–b ) Western blot analysis of STAT3 and NF-κB in AGS cell lines stably expressing TFF1 or empty vector pcDNA infected with H. pylori J166 ( a ) or 7.13 ( b ) strains at different time points 1, 6 and 24 h. H. pylori increases of P-NF-κB-P65 (S536) and P-STAT3 (Y705) protein levels after 1 and 24 h of infection, respectively and reconstitution of TFF1 expression abolished this increase. β-ACTIN was used as a protein loading control, and TFF1 expression was confirmed using TFF1 specific antibody. The relative intensity ratio of p-STAT3 (y705)/β-Actin and p-NF-κB/β-Actin were calculated by the Image-Lab software from BioRad. The Western blot results are representative of three independent experiments. The results are expressed as mean ± SEM of at least three independent experiments using a two-tailed Student’s t -test

Article Snippet: Chromatin solutions were immunoprecipitated with NF-κB (Cat# 8242) or STAT3 (Cat# 12640) monoclonal antibodies from cell signaling (1:100 dilution) at 4 °C overnight.

Techniques: Activation Assay, In Vitro, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Infection, Control, Software, Two Tailed Test

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: TFF1 abolishes H. pylori -induced increase of NF-κB and STAT3 binding to its target gene IL6. a – b ChIP assay in AGS-pcDNA and AGS-TFF1 stable cells infected with H. pylori (7.13) for a period of 4 h for NF-κB binding ( a ) and 24 h for STAT3 binding ( b ), followed by quantitative real-time PCR with primers designed for STAT3 and NF-κB binding site on IL6 promoter regions. Control primers were designed 500 base pair upstream of STAT3 binding site on the IL6 promoter. These primers were used as negative control (NC). Results are presented as a percentage of input

Article Snippet: Chromatin solutions were immunoprecipitated with NF-κB (Cat# 8242) or STAT3 (Cat# 12640) monoclonal antibodies from cell signaling (1:100 dilution) at 4 °C overnight.

Techniques: Binding Assay, Infection, Real-time Polymerase Chain Reaction, Control, Negative Control

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: BAY and Iκ-B super repressor inhibit STAT3 activation. a The luciferase reporter assay using a STAT3-Luc ( a ) reporter plasmids. H. pylori infection of AGS-pcDNA cells significantly increased the luciferase activity, which was reduced after treatment with BAY (10 µM) or transfection with Iκ-B super-repressor plasmid The bar graphs represent the mean ± SEM of 3 independent experiments. b Western blot analysis of p-STAT3 in AGS-pcDNA cell lines infected with H. pylori, 7.13 treated with BAY, or transfected with Iκ-B super repressor (Iκ-BSR). The increase of p-STAT (Y705) protein level in H. pylori -infected cells was abolished after treatment with BAY or transfected with Iκ-BSR plasmid. The results are representative of three independent experiments

Article Snippet: Chromatin solutions were immunoprecipitated with NF-κB (Cat# 8242) or STAT3 (Cat# 12640) monoclonal antibodies from cell signaling (1:100 dilution) at 4 °C overnight.

Techniques: Activation Assay, Luciferase, Reporter Assay, Infection, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: Treatment with STAT3 or NF-κB inhibitors reduced inflammation in TFF1-KO mice gastric tissues. a Immunofluorescence staining of phospho-NF-κB-P65 (Green) and phospho-STAT3 (Y705) (Red) in the antropyloric region of the glandular stomach of the TFF1-KO mice infected with PMSS1 H. pylori and treated or not by intraperitoneal injection with BAY (5 mg/Kg). In control, un-infected TFF1-KO showed more p-NF-κB-P65 and p-STAT3 nuclear staining after H. pylori infection (arrowheads). However, after treatment with BAY, staining showed reduced nuclear STAT3 and NF-κB (arrows). 4’, 6’ Diamino-2-phenylindole (DAPI) (blue) was used as a nuclear counterstain, original magnification (× 1000). b RT-qPCR analysis showing mRNA expression of pro-inflammatory target genes ( Il-6, Vegf-α, Il-17 and IL-23 ) in H. pylori -infected TFF1-KO mice (10–12 weeks of age) treated or not with BAY (5 mg/Kg) and compared to TFF1-KO uninfected mice. Results are graphed using box-and-whisker blots to depict the smallest value, lower quartile, median, upper quartile, and largest value. (●) Indicate the mean. c A schematic cartoon depicting the role of TFF1 in regulating the inflammation in gastric epithelial cell through inhibition of NF-κB-mediated activation of STAT3 in response to H. pylori

Article Snippet: Chromatin solutions were immunoprecipitated with NF-κB (Cat# 8242) or STAT3 (Cat# 12640) monoclonal antibodies from cell signaling (1:100 dilution) at 4 °C overnight.

Techniques: Immunofluorescence, Staining, Infection, Injection, Control, Quantitative RT-PCR, Expressing, Whisker Assay, Inhibition, Activation Assay

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Cardiac-specific Dsg2 knockout induced cardiac fibrosis. ( A ) Masson staining of heart sections in WT and CS-Dsg2 −/− (−/−) mice. Arrow shows cardiac fibrosis. Collagen volume fraction in the hearts of WT and CS-Dsg2 −/− mice was assessed. ( B ) Representative Western blots from mouse left ventricular (LV), interventricular septum (IVS), and right ventricle (RV). DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse LV and RV are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Knock-Out, Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Effects of Dsg2 siRNA and Stat3 siRNA on the expression levels of fibrotic markers in HL-1 cells. ( A , B ) HL-1 cells were transfected with control siRNA or Dsg2 siRNA. ( A ) Representative Western blots for DSG2, pSTAT3, α-SMA, and Collagen I were detected using specific antibodies. STAT3 and β-actin were used as loading controls. ( B ) Results of quantitative PCR analysis of Dsg2, α-SMA, and Collagen I mRNA levels in HL-1 cells treated with control or Dsg2 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01 vs. control. ( C , D ) HL-1 cells were transfected with control siRNA or Stat3 siRNA. ( C ) Representative Western blots for STAT3 and α-SMA were detected using specific antibodies. β-actin were used as loading controls. ( D ) Results of quantitative PCR analysis of α-SMA and Collagen I mRNA levels in HL-1 cells treated with control or Stat3 siRNA are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 3. * p < 0.05, ** p < 0.01, vs. control.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: Fenofibrate alleviated cardiac fibrosis in CS-Dsg2 −/− mice. ( A ) Masson staining of heart sections in WT, CS-Dsg2 −/− mice, and CS-Dsg2 −/− mice treated with fenofibrate (Dsg2 −/− F). Collagen volume fraction in the hearts of WT, CS-Dsg2 −/− , and Dsg2 −/− F mice were assessed. ( B ) Representative Western blots from ventricles of WT, CS-Dsg2 −/− , and Dsg2 −/− F mice. DSG2, PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Cells

Article Title: Activation of PPARα Ameliorates Cardiac Fibrosis in Dsg2-Deficient Arrhythmogenic Cardiomyopathy

doi: 10.3390/cells11203184

Figure Lengend Snippet: AAV9-Pparα alleviated cardiac fibrosis in CS-Dsg2 −/− mice. ( A ) Masson staining of heart sections in CS-Dsg2 −/− mice and CS-Dsg2 −/− mice received AAV9-Pparα (Dsg2 −/− P). Collagen volume fraction in the hearts of CS-Dsg2 −/− and Dsg2 −/− P mice were assessed. ( B ) Representative Western blots from ventricles of CS-Dsg2 −/− and Dsg2 −/− P mice. PPARα, pSTAT3, pSMAD3, pAKT, α-SMA, and Collagen I were detected using specific antibodies. STAT3, SMAD3, AKT, and GAPDH were used as loading controls. ( C ) Results of quantitative PCR analysis of PPARα, TGF-β, α-SMA, and Collagen I mRNA levels in mouse ventricles are expressed as fold change of control using β-actin as loading control. Results are expressed as mean values ± SEM. n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CS-Dsg2 −/− .

Article Snippet: For transient transfection, cells were plated at optimal densities and grown for 24 h. Cells were then transfected with Dsg2 siRNA (MBS828119, MyBioSource, San Diego, CA, USA) or Stat3 siRNA (6354, Cell Signaling Technology) using lipofectamine reagent according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Oncology

Article Title: Combined inhibition of IL-6 and IL-8 pathways suppresses ovarian cancer cell viability and migration and tumor growth

doi: 10.3892/ijo.2022.5340

Figure Lengend Snippet: Baze and SCH inhibit the expression of target genes downstream of the IL-6 and IL-8 pathways in human ovarian cancer cells. SKOV3 and CAOV3 cells were treated with DMSO, a single drug or their combination. The levels of p-STAT3, p-AKT, p-S6 and survivin were determined by western blot analysis. (A) CAOV3. (B) SKOV3. * P<0.05, ** P<0.01, *** P<0.001 and **** P<0.0001 (DMSO vs. Baze, SCH or Baze + SCH). B+S, Baze + SCH; B10, BAZE 10 µ M; B5, BAZE 5 µ M; BAZE, Baze; S50, SCH 50 µ M; p-phosphorylated; SCH, SCH.

Article Snippet: The following primary antibodies were purchased from Cell Signaling Technology, Inc. and diluted at 1:1,000 in 5% milk: Phosphorylated (p)-STAT3 (Y705; rabbit mAb; cat. no. 9131S), STAT3 (rabbit mAb; cat. no. 4904S), p-S6 (rabbit mAb; cat. no. 4858S), S6 (rabbit mAb; cat. no. 2217S), p-AKT (rabbit mAb; cat. no. 4060S), AKT (rabbit mAb; cat. no. 4691S), survivin (rabbit mAb; cat. no. 2808S) and GAPDH (rabbit mAb; cat. no. 2118S).

Techniques: Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 1. Ablation of Stat3 in bone marrow cells gives rise to skewed myeloid cell differentiation and hypersensitivity to GM-CSF. A, morphological, histological, and flow cytometric analyses of spleens from Stat3f/f/btie2-cre and Stat3f/f littermate control mice. Panel a, splenomagaly was found in Stat3f/f/ btie2-cre mice; panel b, comparison of spleen weight to body weight ratio of Stat3f/f and Stat3f/f/btie2-cre mice, p 0.01 (Student’s t test, 2 tails); panels c and d, disrupted spleen architecture in Stat3f/f/btie2-cre mice (d) when compared with Stat3f/f controls (c); panel e, both F480Mac1 and GR-1Mac1 popula- tions were significantly increased in Stat3f/f/btie2-cre spleens compared with the Stat3f/f control spleens. B, bone marrow F480Mac1 and GR-1Mac1 populations were increased in the Stat3f/f/btie2-cre mice compared with Stat3f/f control mice. C, methylcellulose-based colony forming assays demonstrated increased bone marrow progenitor-derived colonies in response to increasing doses of GM-CSF from the Stat3f/f/btie2-cre mice compared with the Stat3f/f mice. D,increasedproliferativeactivityofStat3f/f/btie2-crebonemarrowcellsinresponsetovariouscytokines.Thefinalconcentrationofallcytokines,exceptIL-3(100u/ml), was 30 ng/ml. E, decreased apoptosis in Stat3f/f/btie2-cre bone marrow cells when cultured in a low serum in the presence of 0.1 or 1 ng/ml of GM-CSF. Error bars represent S.D.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Cell Differentiation, Control, Comparison, Derivative Assay, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 2. Shp2 gain-of-function mutants negatively regulate Stat3 acti- vationinmurinebonemarrowcellsandperipheralbloodnucleatedcells from individuals with NS. A, panel a, Western blot analyses examining GM- CSF-stimulated (20 ng/ml) Stat3 activation in the mouse bone marrow-de- rived macrophage progenitors transduced with MIEG3 (empty vector), WT Shp2, or Shp2E76K. Panel b, density quantification of protein band in A, panel a; B, panel a, representative flow cytometric analysis showing reduced Stat3 tyrosine phosphorylation in peripheral blood cells from individuals with NS in both the quiescent state and after IL-6 stimulation. Panel b, statistical analysis

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Western Blot, Activation Assay, Transduction, Plasmid Preparation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 3. Stat3activationinvalvulogenesis,andShp2gain-of-functionmutantsinhibittheactivationofStat3inresponsetoEGF.A,immunohistochemistry staining of activated Stat3 (pY Stat3) in developing and mature PV of wild type mice. Nuclear brown signals are positive staining. Activated Stat3 is apparent in E14.5 embryonic heart and is restricted to the developing leaflets (A, panels a to c), and remains into adult stage (panel d). Activated Stat3 is present in the PV of control embryo hearts at E17.5 (panel e), absent in PV from Stat3f/f/nestin-cre embryo heart at the same stage (panel f). B, panel a, after serum deprivation overnight, Western blotanalysiswasusedtoexaminebasalandEGF-stimulated(50ng/ml)Stat3tyrosinephosphorylationinNIH3T3cellsexpressingShp2mutants(N308DandE76K)or controls(MIEG3orWTShp2).Panelb,densityquantificationoftheproteinbandinB,panela.C,EMSAtoexamineStat3DNAbindingactivityusingthem67-SIEprobe and nuclear extracts from NIH3T3 cells transduced with MIEG3 (M), WT Shp2 (WT), Shp2N308D (N308D), or Shp2E76K (E76K). Error bars represent S.D.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Control, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 4. Conditional deletion of Stat3 with nestin-cre induces pulmonary stenosis due to the thicken- ing of leaflets. A, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside(X-gal) staining of nestin-cre/Rosa26R mouse embryos and newborns indicates the cre activity in the developing mouse embryos. A, panels a and b, central nervous system is positive for cre activity in the mouse embryos at E10.5. Panels c and d, cre activity was found in the pulmonary valves (PV) and the aortic valves (AV) at E14.5 (A, panel c) and P0 (B, panel d). B, ablation of Stat3 with nestin-cre leads to pulmonary stenosis caused by the thickening and enlargement of leaflets. B, panel a, newborn of Stat3f/f (f/f) and Stat3f/f/Nestin-cre (c;f/f) mice. B, panel b, hematoxylin and eosin staining of the transverse section of heart from control (f/f) and STAT3f/f/Nestin-cre (c;f/f) newborn mice (P0). Note that Stat3 ablation leads to right ventricle dilation in Stat3f/f/Nestin-cre mice. B, panels d–g, hematoxylin and eosin staining of sections of the pulmonary valve and aorta valve regions in control (B, panels d and f) and Stat3f/f/ Nestin-cre mutant (B, panels e and g) newborn mice. LV, left ventricle; RV, right ventricle. B, panel h, quantifica- tionofpulmonaryvalvethicknessinStat3f/f/Nestin-creandcontrolhearts(P0)(n6,p0.03,Student’spaired t test; 2 tails).

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Staining, Activity Assay, Control, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 5. Shp2 dephosphorylates Tyr(P)-Stat3 and Shp2-Stat3-medi- atedsignalingcriticallycontributestothepathogenesisofNS.A,Western blot analysis of Stat3-deficient bone marrow-derived macrophage progeni- tors treated with GM-CSF to assess Stat3 and ERK activation. B, panel a, puri- fied constitutively active Shp2 protein (catalytic domain) was capable of dephosphorylating Tyr(P)-Stat3. Stat3 protein was immunoprecipitated from IL-6-treated Raw 264.7 cell extract, and then incubated with purified recom- binant Shp2 protein (active form, 2 g) for the indicated time with or without preincubation of the small molecule Shp2 phosphatase inhibitor IIB-08 (100 M). Panel b, Shp2 inhibitor IIB-08 inhibits ERK activation, whereas enhancing the Tyr(P)-Stat3 level, in IL-6 induced Raw 264.7 cells. Raw 264.7 cells were pretreated with the Shp2 inhibitor IIB-08 (10 M), or dimethyl sulfoxide (DMSO) for 60 min, and then incubated with IL-6 (20 ng/ml) for 60 min before being subjected to lysis and Western blot analysis.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Incubation, Purification, Lysis

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 7. Schematic diagram of dual-signaling pathways regulated by Shp2. In normal physiological conditions, Stat3 activity is under the positive and negative regulation of Janus kinases and Shp2, respectively (A). Shp2 gain-of-function mutations lead to the hyperactivation of the RAS/ERK signaling pathway and the excessive inactivation of Stat3, which synergistically promotes the pathogenesis of Noonan syndrome and/or JMML (B).

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Activity Assay