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

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: Oncotarget

Article Title: Impaired lymphatic function accelerates cancer growth

doi: 10.18632/oncotarget.9953

Figure Lengend Snippet: A. Multiplex ELISA of tumor interstitial fluid from C3HBA tumors demonstrating significantly higher IL-6 level in Chy compared to wt mice. Bars depict the mean cytokine concentration ± SEM, n=8 per group (except GM-CSF: n=3 per group). *p<0.05. B. ELISA of tumor interstitial fluid from KHT-1 tumors demonstrating no significant difference in IL-6 levels between Chy and wt mice. Bars depict the mean cytokine concentration ± SEM, n=5 per group. C. Tumor growth of C3HBA breast cancer in Chy and wt mice treated with either IL-6 receptor antibody (tocilizumab) or placebo (saline) i.p for 21 days. The graphs depict the mean tumor volume ± SEM, n=7 per group. Arrows indicate when the treatment started. Tocilizumab significantly reduced tumor growth both in Chy and wt mice. *p<0.05. D. Western blot analysis demonstrating reduced STAT3 phosphorylation (Ser727) in C3HBA tumors when IL-6 signaling is inhibited with tocilizumab. Densitometry of western blots presented as the ratio of protein of interest normalized to STAT3 and actin expression. Bars depict the mean ± SEM, n=3 per group. *p<0.05

Article Snippet: Rabbit anti-actin (Sigma), rat anti-IL1-beta/IL-1F2 (R&D), rabbit anti-TGF-beta (Nordic Biosite), rat anti-GM-CSF (Abcam), rat anti-IL10 (Abcam), goat anti-M-CSF (Abcam), goat anti-VEGFD (Abcam), rabbit anti-VEGF (Abcam), rabbit anti-VEGFC (Abcam), rabbit anti-PlGF (Abcam), rabbit anti-IL-6 (Abcam), rabbit anti-phospho (Ser727) STAT3 (Cell Signaling), rabbit anti-phospho, (Tyr705) STAT3 (Cell Signaling), rabbit anti-STAT3 (Cell Signaling).

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Saline, Western Blot, Phospho-proteomics, Expressing

Journal: BMJ Open Diabetes Research & Care

Article Title: Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

doi: 10.1136/bmjdrc-2020-001242

Figure Lengend Snippet: JAK/STAT, inflammatory and oxidative stress pathways in kidney tissue of BTBR ob/ob model. (A) Representative images of phosphorylated (p-)STAT3, p-p65 NF-κB and p-NRF2, and quantification of positive cells in glomerular and tubular fields of BTBR WT and ob/ob mice. Magnification ×400 and ×630. Arrows indicate positive stained cells. (B) Representative images of SOCS1/SOCS3 proteins and quantification of positive stained area per tubular field. Magnification ×200. (C) Real-time PCR analysis of JAK/STAT, inflammatory and oxidative stress genes. Values normalized by endogenous control gene 18s are expressed as n-fold of the average value from BTBR WT. Data are shown as scatter dot plots and mean±SD of each group (n=5–7 mice/group); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 versus BTBR WT control. (D) Schematic protein–protein interaction prediction of JAK/STAT, inflammatory and oxidative stress markers in Mus musculus according to STRING software. More information can be found at https://string-db.org/ . a.u., arbitrary units; BTBR, black and tan brachyuric; GCS, glomerular cross section; JAK/STAT, Janus kinase/signal transducers and activators of transcription; NF-κB, nuclear factor-κB; NRF2, nuclear factor erythroid 2-related factor 2; ob/ob, obese/obese; SOCS, suppressor of cytokine signaling; WT, wild type.

Article Snippet: The primary antibodies for immunodetection were sourced as follows: phosphorylated (p-) STAT3 serine 727 (Cell Signaling Technology Cat# 9134, RRID:AB_331589, dilution 1:100), p-STAT1 tyrosine 701 (Cell Signaling Technology Cat# 7649, RRID:AB_10950970, dilution 1:50), p-p65 subunit of nuclear factor-κB (NF-κB) serine 536 (Santa Cruz Biotechnology Cat# sc-33020, RRID:AB_2179018, dilution 1:100), p-nuclear factor erythroid 2-related factor 2 (NRF2) serine 40 (Abcam Cat# ab76026, RRID:AB_1524049, dilution 1:2000), SOCS1 (Abcam Cat# ab62584, RRID:AB_956316, dilution 1:1000), SOCS3 (Abcam Cat# ab16030, RRID:AB_443287, dilution 1:200), F4/80 monocytes/macrophages (Bio-Rad Cat# MCA497, RRID:AB_2098196, dilution 1:70), CD3 T lymphocytes (Agilent Cat# M7254, RRID:AB_2631163, dilution 1:100), Wilms tumor protein-1 (WT-1; Agilent Cat# M3561, RRID:AB_2304486, dilution 1:100), perilipin-1 (sc-390169, dilution 1:50, Santa Cruz Biotechnology, USA) and 4-hydroxy-2-nonenal (4-HNE; Abcam Cat# ab46545, RRID:AB_722490, dilution 1:200).

Techniques: Staining, Real-time Polymerase Chain Reaction, Control, Software

Journal: BMJ Open Diabetes Research & Care

Article Title: Anti-inflammatory, antioxidant and renoprotective effects of SOCS1 mimetic peptide in the BTBR ob/ob mouse model of type 2 diabetes

doi: 10.1136/bmjdrc-2020-001242

Figure Lengend Snippet: MiS1 treatment inhibits kidney JAK/STAT activation and renal microinflammatory milieu in the BTBR ob/ob model. (A) Graphs and images represent the changes observed in JAK/STAT activation (p-STATs) in diabetic mice treated with active MiS1 (2 µg and 4 µg) and inactive mutated peptide (Mut 4 µg) compared with vehicle controls (Veh), quantified per number of positive cells p-STAT1+ and p-STAT3+, both at the glomerular and tubular fields. Magnification ×630. (B) Representative images of immunohistochemistry against F4/80 and CD3. Magnification ×200 and ×630. Graphs represent the quantification of average number of monocytes/macrophages F4/80+ and CD3+ T lymphocytes, both at the glomerular and interstitial fields. Arrows indicate positively stained cells. (C) Gene expression analysis of mRNA related with JAK/STAT pathway ( Stat1 , Stat3 , Socs1 and Socs3 ), inflammatory cytokines ( Tnfα and Il-12 ) and chemokines ( Mcp-1 and Rantes ), and kidney damage markers ( Kim-1 and Ngal ) were evaluated by real-time PCR, being normalized in each sample by endogenous control gene 18s and expressed as n-fold the average value obtained in the vehicle group (Veh). Data are shown as scatter dot plots and mean±SD of each group (n=6–7 mice/group); *p<0.05, **p<0.01, ****p<0.0001 versus diabetic vehicle control. BTBR, black and tan brachyuric; GCS, glomerular cross section; JAK/STAT, Janus kinase/signal transducers and activators of transcription; ob/ob, obese/obese.

Article Snippet: The primary antibodies for immunodetection were sourced as follows: phosphorylated (p-) STAT3 serine 727 (Cell Signaling Technology Cat# 9134, RRID:AB_331589, dilution 1:100), p-STAT1 tyrosine 701 (Cell Signaling Technology Cat# 7649, RRID:AB_10950970, dilution 1:50), p-p65 subunit of nuclear factor-κB (NF-κB) serine 536 (Santa Cruz Biotechnology Cat# sc-33020, RRID:AB_2179018, dilution 1:100), p-nuclear factor erythroid 2-related factor 2 (NRF2) serine 40 (Abcam Cat# ab76026, RRID:AB_1524049, dilution 1:2000), SOCS1 (Abcam Cat# ab62584, RRID:AB_956316, dilution 1:1000), SOCS3 (Abcam Cat# ab16030, RRID:AB_443287, dilution 1:200), F4/80 monocytes/macrophages (Bio-Rad Cat# MCA497, RRID:AB_2098196, dilution 1:70), CD3 T lymphocytes (Agilent Cat# M7254, RRID:AB_2631163, dilution 1:100), Wilms tumor protein-1 (WT-1; Agilent Cat# M3561, RRID:AB_2304486, dilution 1:100), perilipin-1 (sc-390169, dilution 1:50, Santa Cruz Biotechnology, USA) and 4-hydroxy-2-nonenal (4-HNE; Abcam Cat# ab46545, RRID:AB_722490, dilution 1:200).

Techniques: Activation Assay, Immunohistochemistry, Staining, Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: Neoplasma

Article Title: Histone deacetylase inhibitors in plasma cell leukemia treatment: effect of bone marrow microenvironment.

doi: 10.4149/neo_2017_209

Figure Lengend Snippet: Figure 4. Effect of VPA and SAHA on STAT3 phosphorylation. pSTAT3 (Tyr705) level was measured by means of ELISA in UHKT-944 cells. Cells were co-cultured with or without BMSCs and incubated with indicated concentrations of SAHA and VPA for 48 hours. Data represent mean ± standard deviation of three independent experiments. Differences were not statistically significant, P>0.05.

Article Snippet: Phosphorylated STAT3 level was measured after 48 hours treatment with SAHA and VPA using PathScan® PhosphoStat3 (Tyr705) Sandwich ELISA kit (Cell Signaling) following manufacturer ́s instructions.

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Standard Deviation