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:

Article Title: Pre-clinical development of a bi-functional cancer cell homing, PKC? inhibitory peptide for the treatment of head and neck cancer

doi: 10.1158/0008-5472.CAN-08-3465

Figure Lengend Snippet: HN1-PKCε retards tumor growth in a pre-clinical model of HNSCC. A. Bioluminescence images from a representative mouse in the untreated arm, HN1-control treatment arm, and HN1-PKCε treatment arm. B. Bioluminescence. Tumor bioluminescence activity was measured using the Xenogen IVIS Spectrum Imaging System. * p-value < 0.02. C. Tumor proliferation and apoptotic index. Tumor sections were stained for proliferating cells using an anti-Ki67 antibody (DAKO, Denmark) and apoptotic cells using the ApopTag kit (Intergen Company, Purchase, NY). Proliferation and apoptotic index was determined by counting the number of Ki67- and TUNEL-positive cells per 500 total cells in five separate random fields at high power (400X). * p-value < 0.001. D. Tumor pSTAT (S727) levels. Tumor section were stained for S727-phosphorylated Stat3 using an anti-phospho-S727-Stat3 antibody (Cell Signaling, Beverly, MA). These are representative sections for each treatment arm.

Article Snippet: Tumor section were stained for S727-phosphorylated Stat3 using an anti-phospho-S727-Stat3 antibody (Cell Signaling, Beverly, MA).

Techniques: Activity Assay, Imaging, Staining, TUNEL Assay

Journal: International Journal of Biological Sciences

Article Title: Chemical and genetic inhibition of STAT3 sensitizes hepatocellular carcinoma cells to sorafenib induced cell death

doi: 10.7150/ijbs.22220

Figure Lengend Snippet: JAK inhibitor I enhanced sorafenib induced cell death in STAT3 active HCC cells. (A, C, E) HCC cells were treated with 2uM JAK inhibitor I (JAKi), 5uM Sorafenib or their combination. The expression levels of STAT3, phospho-STAT3 (Y705) phospho-STAT3 (S727), Mcl-1 and GAPDH were determined by Western Blot. (B, D, F) Cell death assays were performed by pI staining and photographed under microscopy. Cell death rate was calculated by Image J. Data of three independent replicates are presented as the mean+/-s.e.m., n=3. (G) Huh7 cells were treated with JAKi (2uM), Sorafenib (5uM) and their combination for 60hours. Cell death assay was performed by pI staining and followed by flow cytometry analysis.

Article Snippet: Antibodies of anti STAT3 (#9139), anti phospho-STAT3 at Y705 (#9145) or anti phospho-STAT3 at S727 (#94994) were from Cell Signaling (Davers, MA).

Techniques: Expressing, Western Blot, Staining, Microscopy, Flow Cytometry

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

Journal: Journal of Biological Chemistry

Article Title: Interferon α-induced Apoptosis in Tumor Cells Is Mediated through the Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Signaling Pathway

doi: 10.1074/jbc.m312219200

Figure Lengend Snippet: FIG. 4. STAT phosphorylation and activation are not affected by the inhibition of the PI3K or mTOR. U266 cells were treated with 5000 units/ml of IFN alone or together with Ly294002 or rapamycin for the indicated times. A, immunoblots for the phosphorylation specific sites of STAT3, STAT1, and STAT2 were performed. The same membranes were reprobed with anti-tubulin antibodies as a control for loading. B, cells were cytospun onto glass slides, fixed, and stained with rabbit serum against phosphotyrosine-STAT3, and detected by using fluorescein isothiocyanate-conjugated secondary antibody. The nuclei were stained with DAPI. DMSO, dimethyl sulfoxide. C, nuclear extracts from the indicated samples were incubated with 32P-labeled ISRE (top panel) or SIE (bottom panel) double-stranded oligonucleotides followed by EMSA.

Article Snippet: The following antibodies were used: rabbit polyclonal IgG against phospho-STAT1 (Y701 and S727), phospho-STAT2 (Y689), phospho-STAT3 (Y705 and S727), phospho-Akt (S473 and T308), phospho-p70S6K (T389 and T421/S424), phospho-4EBP1 (T37/46), and total 4E-BP1 from Cell Signaling Technology; mouse monoclonal anti-IRF-9 (ISGF3 ) from Transduction Laboratories; rabbit IgG against IRF-1 (C-20) from Santa Cruz Biotechnology, Inc.; anti-MxA (myxovirus resistance protein A) chicken serum from Baclab (Pratteln); and HRP-conjugated anti-chicken from Sigma.

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Western Blot, Control, Staining, Incubation, Labeling

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A) The transfection efficiency of the siRNA was analysed in OS cells at different time points after transfection using flow cytometry. (B) Western blot analysis of STAT3 and pSTAT3 expression in OS cells treated with ADSC-conditioned medium. OS cells were pretreated with the STAT3 siRNA (50 nM) for 48 h before treatment with ADSC-conditioned medium for an additional 12 h. STAT3 expression levels were detected by western blotting. OS cells treated with Lipofectamine 2000 alone served as negative controls. (C) OS cell invasion was analysed using transwell assay. OS cells were transfected with the siRNA and treated with ADSC-conditioned medium. OS cells treated with ADSC-conditioned medium alone served as positive controls, and OS cells treated with Lipofectamine 2000 alone served as negative controls. (D) The MMP2 and MMP9 levels in the supernatants were detected using ELISAs. (E) OS cells were treated with the STAT3 siRNA or ADSC-conditioned medium, and MMP2/9 and E-cadherin expression in OS cells was examined by western blotting. OS cells treated with Lipofectamine 2000 alone served as negative controls. The results are expressed as mean±SD. Abbreviation: C Control; L Lipofectamine 2000; S siRNA. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: Transfection, Flow Cytometry, Western Blot, Expressing, Transwell Assay, Control

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A-B) Cell proliferation was evaluated using the CCK8 assay and EdU analysis. OS cells were transfected with the siRNA and treated with ADSC-conditioned medium. OS cells treated with ADSC-conditioned medium alone served as positive controls, and OS cells treated with Lipofectamine 2000 alone served as negative controls. (C-D) OS cells were transfected with the STAT3 siRNA or treated with ADSC-conditioned medium, and apoptosis rates were determined using flow cytometry. OS cells treated with Lipofectamine 2000 alone served as negative controls. The percentages of Annexin V-positive cells are presented in bar charts. *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: CCK-8 Assay, Transfection, Flow Cytometry

Journal: Oncotarget

Article Title: Adipose-derived mesenchymal stem cells promote osteosarcoma proliferation and metastasis by activating the STAT3 pathway

doi: 10.18632/oncotarget.15866

Figure Lengend Snippet: (A) An in vivo imaging system was used to monitor OS xenograft luminescence activity, which represented tumour growth and metastasis. (B) Living Image Software was used to analyse tumour bioluminescence intensity weekly. The quantitation of the normalized image counts is shown. (C) Lungs of the tumour-bearing mice were excised, and the bioluminescence intensity was analysed to determine the level of tumour metastasis in the lungs. (D) Survival curves of the three groups are shown, and the median survival of the OS group was 43 days, which was significantly longer than the survival of the OS + conditioned-medium group (25 days, P<0.01). (E) The immunohistochemical analysis of Ki67, STAT3, MMP2 and MMP9 expression in the orthotopic tumour xenografts is shown. (F) Quantitation of the intensity of Ki67, STAT3 and MMP2/9 staining in the xenografts. Scale bar: 25 μm. * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Antibody staining with 100 μL of specific antibodies against human STAT3 (1:100 dilution; R&D Systems) and rabbit monoclonal antibodies against human Ki67, MMP2 and MMP9 (1:100 dilution; ProteinTech groups inc, Chicago, IL) was applied to the cells, which were incubated overnight at 4°C.

Techniques: In Vivo Imaging, Activity Assay, Software, Quantitation Assay, Immunohistochemical staining, Expressing, Staining

Journal: Aging (Albany NY)

Article Title: SLCO1B3 promotes colorectal cancer tumorigenesis and metastasis through STAT3

doi: 10.18632/aging.203502

Figure Lengend Snippet: Bioinformatics analysis of GSE163396. Microarray data analysis was performed to investigate the molecular mechanisms underlying the function of SLCO1B3 . ( A ) Heatmap of 286 differentially expressed genes from GSE163396 dataset (with 125 highly expressed genes and 161 lowly expressed genes). ( B – D ) GO functional enrichment and KEGG pathway analysis were performed based on DEGs from GSE163396 dataset. Partial results of the upregulated GO pathways were shown in panel B , the downregulated GO pathways were shown in panel C , and the KEGG pathway was illustrated in panel D . ( E ) Gene Set Enrichment Analysis (GSEA) revealed that most DEGs associated with the STAT3 signaling pathway were enriched in the SLCO1B3 gene. ( F ) The co-expression analysis revealed a positive association between the SLCO1B3 and STAT3 activation.

Article Snippet: The primary antibodies used in the experiments included an anti- OATP1B3 antibody from Proteintech Group Inc., (Wuhan, China; Catalog # 66381-1) and anti-STAT3 (Catalog # ab68153), anti-p-STAT3 (S727) (Catalog # ab32143), anti-MMP-2 (Catalog # ab92536), and anti-MMP9 (Catalog # ab58803) antibodies from Abcam (USA).

Techniques: Microarray, Functional Assay, Expressing, Activation Assay

Journal: Aging (Albany NY)

Article Title: SLCO1B3 promotes colorectal cancer tumorigenesis and metastasis through STAT3

doi: 10.18632/aging.203502

Figure Lengend Snippet: Ct-SLCO1B3 knockdown in HCT116 cells downregulates p-STAT3, MMP-2, and MMP-9. HCT116 cells were transiently transfected with si1-Ct-SLCO1B3 , si2-Ct-SLCO1B3 , or si-NC with or without IL-6 stimulation (50 ng/mL). The protein levels of Ct-OATP1B3 , p-STAT3, total STAT3, MMP-2, and MMP-9 were determined by western blot analysis. ( A ) Gel image. ( B ) Quantified protein levels without IL-6 stimulation. n=3, **P < 0.01 vs. si-NC. NC=si-NC, Si-1= si1-Ct-SLCO1B3 , Si-2= si2-Ct-SLCO1B3 .

Article Snippet: The primary antibodies used in the experiments included an anti- OATP1B3 antibody from Proteintech Group Inc., (Wuhan, China; Catalog # 66381-1) and anti-STAT3 (Catalog # ab68153), anti-p-STAT3 (S727) (Catalog # ab32143), anti-MMP-2 (Catalog # ab92536), and anti-MMP9 (Catalog # ab58803) antibodies from Abcam (USA).

Techniques: Transfection, Western Blot

Journal: Aging (Albany NY)

Article Title: SLCO1B3 promotes colorectal cancer tumorigenesis and metastasis through STAT3

doi: 10.18632/aging.203502

Figure Lengend Snippet: The effects of Ct-SLCO1B3 knockdown on CRC metastasis in vivo . ( A , B ) H&E staining of the liver and lung tissues showing metastatic nodules. ( C ) The expression of p-STAT3, STAT3, MMP-2, and MMP-9 in metastatic tumors by western blot analysis. **P < 0.01 vs. sh-Control.

Article Snippet: The primary antibodies used in the experiments included an anti- OATP1B3 antibody from Proteintech Group Inc., (Wuhan, China; Catalog # 66381-1) and anti-STAT3 (Catalog # ab68153), anti-p-STAT3 (S727) (Catalog # ab32143), anti-MMP-2 (Catalog # ab92536), and anti-MMP9 (Catalog # ab58803) antibodies from Abcam (USA).

Techniques: In Vivo, Staining, Expressing, Western Blot

Journal: Membranes

Article Title: Natural Killer Cell Derived Microvesicles Affect the Function of Trophoblast Cells

doi: 10.3390/membranes13020213

Figure Lengend Snippet: Effect of NK-92 cell MVs on content of STAT1 and STAT3 proteins and their phosphorylated forms in JEG-3 cell lysates. Immunoblot shows content of ( A ) STAT3 and ( E ) STAT1 in intact JEG-3 cells and after their interaction with NK-92 cell MVs. Band density of ( B ) total STAT3 and ( F ) STAT1 content in intact JEG-3 cells and after interaction with NK-92 cell MVs, normalized by GAPDH. Ratio of ( C , D ) phospho-STAT3 (pSTAT3(Ser727), pSTAT3(Tyr(705)) and total STAT3 (n = 3) and ( G , H ) phospho-STAT1 (pSTAT1(Ser727), pSTAT1(Tyr(701)) and total STAT1 (n = 4) in studied samples. Significant differences between JEG-3 cells treated with NK-92 cell MVs and intact JEG-3 cells: * p < 0.05.

Article Snippet: The membranes were incubated overnight at 4 °C with primary monoclonal antibodies, at the indicated dilutions, against STAT3 (rabbit mAb 1:1000), phospho-STAT3 (S727) (mouse mAb 1:1000), phospho-STAT3 (Y705) (mouse mAb 1:1000), ERK1/2 (p44/42 MAPK (ERK1/2), mouse mAb 1:2000), phospho-ERK1/2 (phospho-p44/42 MAPK, rabbit mAb 1:1000), STAT1 (rabbit mAb, 1:1000), phospho-STAT1 (Ser727) (rabbit mAb 1:1000), phospho-STAT1 (Tyr701) (rabbit mAb 1:1000) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot

Journal: Membranes

Article Title: Natural Killer Cell Derived Microvesicles Affect the Function of Trophoblast Cells

doi: 10.3390/membranes13020213

Figure Lengend Snippet: Some molecules included in the composition of NK-92 cell MVs (according to mass spectrometry data [ 83 , 104 ]) and possible signal transduction pathways that involve the phosphorylation of intracellular signaling molecules.

Article Snippet: The membranes were incubated overnight at 4 °C with primary monoclonal antibodies, at the indicated dilutions, against STAT3 (rabbit mAb 1:1000), phospho-STAT3 (S727) (mouse mAb 1:1000), phospho-STAT3 (Y705) (mouse mAb 1:1000), ERK1/2 (p44/42 MAPK (ERK1/2), mouse mAb 1:2000), phospho-ERK1/2 (phospho-p44/42 MAPK, rabbit mAb 1:1000), STAT1 (rabbit mAb, 1:1000), phospho-STAT1 (Ser727) (rabbit mAb 1:1000), phospho-STAT1 (Tyr701) (rabbit mAb 1:1000) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Mass Spectrometry, Transduction, Phospho-proteomics, Migration, Activity Assay, Expressing

Journal: Veterinary Research Communications

Article Title: Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis

doi: 10.1007/s11259-011-9489-3

Figure Lengend Snippet: The number of Gr1-positive cells and p-STAT3 expression in canine mammary non-metastatic adenomas and adenocarcinomas of the 1st, 2nd and 3rd grade of malignancy and in the group of tumors that gave local or distal metastases. Results are presented as means±SD

Article Snippet: After 30 min incubation in 5% bovine serum albumin (Sigma Aldrich, Germany), the following primary antibodies were used (diluted in 1% bovine serum): rat monoclonal anti-Gr1 (in case of paraffin slides) and rabbit polyclonal STAT3 (phospho S727) (for paraffin slides and cell lines) both obtained from Abcam (United Kingdom).

Techniques: Expressing

Journal: Veterinary Research Communications

Article Title: Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis

doi: 10.1007/s11259-011-9489-3

Figure Lengend Snippet: a Pictures of p-STAT3 in canine mammary adenomas, carcinomas of the 1st, 2nd, 3rd grade of malignancy and tumors that gave local/distal metastases ( n = 50) obtained using Olympus BX60 microscope (at the magnification of 200x). The p-STAT3 is reflected as brown color. b The graph of mean optical density of p-STAT3 (and SD) in canine mammary non-metastatic adenomas, carcinomas and metastatic carcinomas. Ten to 20 pictures in each slide were analyzed (three consecutive sections). The colorimetric intensity of the IHC-stained antigen spots was counted by a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA) and the antigen spot color intensity is expressed as mean pixel optical density on a 1–256 scale. The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). The ANOVA and Tukey HSD (Honestly Significant Difference) post-hoc tests were applied to analyze the optical density in all the examined tumor groups. p < 0.001 was regarded as highly significant and marked as ***

Article Snippet: After 30 min incubation in 5% bovine serum albumin (Sigma Aldrich, Germany), the following primary antibodies were used (diluted in 1% bovine serum): rat monoclonal anti-Gr1 (in case of paraffin slides) and rabbit polyclonal STAT3 (phospho S727) (for paraffin slides and cell lines) both obtained from Abcam (United Kingdom).

Techniques: Microscopy, Staining, Software

Journal: Veterinary Research Communications

Article Title: Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis

doi: 10.1007/s11259-011-9489-3

Figure Lengend Snippet: a Representative picture of p-STAT3 in non-metastatic canine mammary cancer cell line (CMT-U309) obtained using Olympus BX60 microscope (at the magnification of 200x). The p-STAT3 is reflected as brown color. b Representative picture of p-STAT3 in canine mammary cell line (CMT-W1) with high metastatic potential obtained using Olympus BX60 microscope (at the magnification of 200x). The p-STAT3 is reflected as brown color. c The graph of mean optical density of p-STAT3 (and SD) in canine mammary non-metastatic (CMT-U27, CMT-U309, P114 and PL20) and metastatic (CMT-W1 and CMT-W2) cancer cell lines. Ten pictures in each slide were analyzed. The colorimetric intensity of the IHC-stained antigen spots was counted by a computer-assisted image analyzer (Olympus Microimage™ Image Analysis, software version 4.0 for Windows, USA) and the antigen spot color intensity is expressed as mean pixel optical density on a 1–256 scale. The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). The unpaired t-test was applied to analyze the optical density in non-metastatic versus metastatic cell lines. p < 0.001 was regarded as highly significant and marked as ***

Article Snippet: After 30 min incubation in 5% bovine serum albumin (Sigma Aldrich, Germany), the following primary antibodies were used (diluted in 1% bovine serum): rat monoclonal anti-Gr1 (in case of paraffin slides) and rabbit polyclonal STAT3 (phospho S727) (for paraffin slides and cell lines) both obtained from Abcam (United Kingdom).

Techniques: Microscopy, Staining, Software

Journal: Veterinary Research Communications

Article Title: Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis

doi: 10.1007/s11259-011-9489-3

Figure Lengend Snippet: The scheme of possible interactions between the up-regulated genes in canine mammary metastatic cell lines, STAT3 and hematopoietic cells. The names of the identified in microarray study genes and STAT3 were bolded and written in italic. The arrows indicate the activation. The bar-headed line indicates inhibition. The explanation of abbreviations: Gr1+ cells – myeloid precursor cells that express Gr1 antigen; MQ – macrophages; NRP – neuropilin receptor; p38MAPK – p38 mitogen activated protein kinase; p53 – p53 gene; SEMA3B – semaphorin 3B; STAT3 – Signal transducer and activator of transcription 3; VEGF – vascular endothelial growth factor; VEGFR – vascular endothelial growth factor receptor. The graph was made in Corel Photo Paint 3.0 (by Corel Draw, USA)

Article Snippet: After 30 min incubation in 5% bovine serum albumin (Sigma Aldrich, Germany), the following primary antibodies were used (diluted in 1% bovine serum): rat monoclonal anti-Gr1 (in case of paraffin slides) and rabbit polyclonal STAT3 (phospho S727) (for paraffin slides and cell lines) both obtained from Abcam (United Kingdom).

Techniques: Microarray, Activation Assay, Inhibition

Journal: Kidney International Reports

Article Title: Inhibition of STAT3 Signaling Reduces IgA1 Autoantigen Production in IgA Nephropathy

doi: 10.1016/j.ekir.2017.07.002

Figure Lengend Snippet: Interleukin-6 (IL-6)−induced STAT3 phosphorylation in IgA1-secreting cells. (a) Time course of IL-6−induced STAT3 phosphorylation (Y705 and S727) from IgA1-secreting cell lines derived from Epstein-Barr virus−immortalized peripheral blood mononuclear cells of 1 healthy control subject (HC) and 1 IgA nephropathy (IgAN) patient. Total STAT3 protein served as load control. This experiment was repeated using IgA1-producing cell lines from 3 IgAN patients and 3 HCs. Only a representative example is shown. (b,c) Densitometric analysis of P-STAT3 (Y705 and S727) levels relative to that of total STAT3 for blot in (a). (d) STAT3 phosphorylation (Y705) in IgA1-secreting cells from 3 HCs and 3 IgAN patients incubated for 15 minutes with or without IL-6. (e) Densitometric analysis of P-STAT3 (Y705) levels relative to that of total STAT3 for blot in (d). Representative blots are shown in (a) and (d) and mean values + SD from 1 experiment with 3 samples in each group are shown in (e).

Article Snippet: After transfer, the membranes were blocked by Superblock (Thermo Fisher Scientific) and incubated with phospho-Y705-STAT3- or phospho-S727-STAT3−specific rabbit polyclonal antibodies, both diluted 1:800 in blocking buffer, or with STAT3-specific mouse monoclonal antibodies diluted 1:10,000 (R & D Systems, Minneapolis, MN).

Techniques: Phospho-proteomics, Derivative Assay, Virus, Control, Incubation