Journal: Frontiers in Oncology

Article Title: 5-Methoxytryptophan Sensitizing Head and Neck Squamous Carcinoma Cell to Cisplatitn Through Inhibiting Signal Transducer and Activator of Transcription 3 (STAT3)

doi: 10.3389/fonc.2022.834941

Figure Lengend Snippet: Inhibitory effect of the combination treatment of Cisplatin (CDDP) and 5-methoxytryptophan (5-MTP) on proliferation involved in the STAT3 signaling pathway. SCC4 and SCC25 cells were treated with CDDP, 5-MTP, or both at indicated concentrations for 6 h (A, B) or 24 h (C, D) . Total cell lysates were extracted at the indicated time points and subjected to western blot with relevant antibodies. GAPDH served as an equal loading control in western blot. Asterisks indicate significant difference in drug-treated cells at indicated concentrations singly in comparison with DMSO-treated cells. *P < 0.05.

Article Snippet: The target proteins were probed with specific antibodies against p-STAT3 (ABclonal; 1:1000, AP0705), p-Akt (ABclonal; 1:1000, AP0637), p-p65 (Cell Signaling; 1:1000, 3033T), p-p38 (ABclonal; 1:1000, AP0526), PCNA (GeneTex; 1:3000, GTX100539), GAPDH (GeneTex; 1:10000, GTX100118).

Techniques: Western Blot, Control, Comparison

Journal: Frontiers in Oncology

Article Title: 5-Methoxytryptophan Sensitizing Head and Neck Squamous Carcinoma Cell to Cisplatitn Through Inhibiting Signal Transducer and Activator of Transcription 3 (STAT3)

doi: 10.3389/fonc.2022.834941

Figure Lengend Snippet: Additional inhibitory effect of the combination treatment of Cisplatin (CDDP) and 5-methoxytryptophan (5-MTP) in 4NQO-induced tongue oral squamous cell carcinoma (OSCC) mouse model. (A) Morphological observation of the DMSO- or drug-treated group in 4NQO-induced cancer mouse model. (B) Digital outlines of representative tongues of DMSO- or drug-treated mice. Asterisks indicate significant difference in drug-treated group singly in comparison with 4NQO-treated group. *P < 0.05 (C) hematoxylin–eosin or immunohistochemical staining with PCNA and p-STAT3 antibodies of tongue cancer with DMSO or drug treatment.

Article Snippet: The target proteins were probed with specific antibodies against p-STAT3 (ABclonal; 1:1000, AP0705), p-Akt (ABclonal; 1:1000, AP0637), p-p65 (Cell Signaling; 1:1000, 3033T), p-p38 (ABclonal; 1:1000, AP0526), PCNA (GeneTex; 1:3000, GTX100539), GAPDH (GeneTex; 1:10000, GTX100118).

Techniques: Comparison, Immunohistochemical staining, Staining

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: Frontiers in Cellular and Infection Microbiology

Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

doi: 10.3389/fcimb.2017.00140

Figure Lengend Snippet: Oligonucleotide sequences used in the quantitative reverse transcription polymerase chain reaction (qRT-PCR) .

Article Snippet: For intracellular staining, a cytofix/cytoperm kit was used (Becton Dickinson) with fluorescently labeled antibodies against transcription factors: Alexa Fluor®488 Mouse Anti-Stat3 (pY705; BD Biosciences) and PE-IRF4 Monoclonal Antibody (3E4)(eBioscience).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

doi: 10.3389/fcimb.2017.00140

Figure Lengend Snippet: Activation of Th17 signaling pathway in CD4+ naïve lymphocyte depends on gingipain activity . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM). After 6 h, CD4+ naïve lymphocytes were added and co-stimulated for 3 consecutive days. At day 1, 2, and 3 after co-incubation, cells were collected and lysed with TRIzol, RNA was isolated and reverse transcriptase PCR was performed. Relative expression of cytokine genes IL-6R, IL-1R, TGF β R, RORc, ROR α , STAT3, RUNX1, IRF4, BATF to the reference house-keeping gene EF2 was measured by Real-Time PCR. Data represent fold increase in expression compared to control levels, which were arbitrarily set at 1 and were analyzed with a Student's t -test ( # P < 0.05, ## P < 0.01, ### P < 0.001 vs. control, * P < 0.05, ** P < 0.01, *** P < 0.001 to P. gingivalis treated cells).

Article Snippet: For intracellular staining, a cytofix/cytoperm kit was used (Becton Dickinson) with fluorescently labeled antibodies against transcription factors: Alexa Fluor®488 Mouse Anti-Stat3 (pY705; BD Biosciences) and PE-IRF4 Monoclonal Antibody (3E4)(eBioscience).

Techniques: Activation Assay, Activity Assay, Derivative Assay, Concentration Assay, Incubation, Isolation, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

doi: 10.3389/fcimb.2017.00140

Figure Lengend Snippet: Activation of STAT3 and IRF4 is dependent on gingipain activity . At the end of each period, T cells were stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. Cells were stained for intracellular (A) IRF4 and (B) pSTAT3 (pY705), and IRF4, and analyzed by flow cytometry. Data show fold increase of the transcription factor positive population, relative to the appropriate control set at 1. Data are presented as a mean ± standard deviation of triplicate assays and were analyzed with a Student's t -test ( # P < 0.05, ## P < 0.01, ### P < 0.001 vs. control, * P < 0.05, ** P < 0.01, *** P < 0.001 to P. gingivalis treated cells).

Article Snippet: For intracellular staining, a cytofix/cytoperm kit was used (Becton Dickinson) with fluorescently labeled antibodies against transcription factors: Alexa Fluor®488 Mouse Anti-Stat3 (pY705; BD Biosciences) and PE-IRF4 Monoclonal Antibody (3E4)(eBioscience).

Techniques: Activation Assay, Activity Assay, Staining, Flow Cytometry, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Traditional Chinese medicine, Fuzheng Kang-Ai decoction, inhibits metastasis of lung cancer cells through the STAT3/MMP9 pathway

doi: 10.3892/mmr.2017.6905

Figure Lengend Snippet: STAT3 regulates MMP9 activity in lung cancer cells treated with FZKA. (A) Protein expression levels of STAT3 were reduced following treatment with FZKA (2 mg/ml; 0, 0.5, 2, 4 and 8 h). *P<0.05 vs. 0 h. (B) To overexpress STAT3, cells (H1650, A549 and PC9) were seeded into 6-well plates, and transfected with pCMV6-AC (negative control) and pCMV6-AC-STAT3 DNA constructs, prior to treatment with FZKA. STAT3 protein expression was then measured by western blot analysis. (C) MMP9 activity was increased by STAT3 overexpression. Following treatment with FZKA, the FZKA-mediated inhibition of MMP9 activity was significantly suppressed by STAT3 overexpression. *P<0.05 and **P<0.01 vs. control (Ctrl). FZKA, Fuzheng Kang-Ai; MMP9, matrix metalloproteinase 9; STAT5, signal transducer and activator of signaling 3; Ctrl, control.

Article Snippet: Membranes were blocked with 5% non-fat milk in TBS containing 1% Tween-20 and were then incubated with primary antibodies against STAT3 (cat. no. 8232; CST Biological Reagents Company Limited; dilution, 1:1,000), p-STAT3 (cat. no. 4093; CST Biological Reagents Company Limited; dilution, 1:1,000), vimentin (cat. no. 12826; CST Biological Reagents Company Limited; dilution, 1:1,000), N-cadherin (cat. no. 13116; CST Biological Reagents Company Limited; dilution, 1:1,000), MMP9 (cat. no. 13667; CST Biological Reagents Company Limited; dilution, 1:1,000) and GAPDH (cat. no. 5174; CST Biological Reagents Company Limited; dilution, 1:3,000) at 4°C overnight.

Techniques: Activity Assay, Expressing, Transfection, Negative Control, Construct, Western Blot, Over Expression, Inhibition, Control

Journal: Scientific Reports

Article Title: In vitro biomaterial priming of human mesenchymal stromal/stem cells : implication of the Src/JAK/STAT3 pathway in vasculogenic mimicry

doi: 10.1038/s41598-024-72862-6

Figure Lengend Snippet: Transcriptomic analysis reveals JAK/STAT signaling pathway as a hub in MSC capillary-like structure formation. ( A) Indirect target proteins of STAT3 were retrieved from STRING as described in the section. ( B) Human mesenchymal stromal/stem cells (MSC) were seeded as either monolayers (Plastic, white bars) or as capillary-like structures on top of Cultrex (Cultrex, black bars) for 4 h. Total RNA was extracted from triplicate samples for each condition, and gene expression modulation was assessed by RT-qPCR as described in the section. ( C) Lysates were isolated from monolayers and from VM cultures, and used to perform immunoblotting analysis of the indicated biomarkers at the protein level. Cropped blots are presented for the sake of clarity and conciseness of data presentation. Uncropped full-length blots are presented in the Supplementary data section, as Supplementary Figure S3C. Protein data are representative from two distinct experiments. RT-qPCR data originate from triplicate sample analysis.

Article Snippet: The polyclonal antibodies against STAT3 (12640 S), Snail (3879 S), FOXC2 (12974 S), and Fibronectin (30903 S), as well as the monoclonal antibody against GAPDH (D4C6R) were all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Isolation, Western Blot

Journal: Scientific Reports

Article Title: In vitro biomaterial priming of human mesenchymal stromal/stem cells : implication of the Src/JAK/STAT3 pathway in vasculogenic mimicry

doi: 10.1038/s41598-024-72862-6

Figure Lengend Snippet: STAT3 pharmacological targeting alters MSC capillary-like structure formation and nuclear expression of FOXC2 and SNAIL. ( A) Human mesenchymal stromal/stem cells (MSC) were seeded on top of Cultrex and capillary-like structures generated for 4 h in the absence (Vehicle) or presence of 10 µM JAK/STAT3 inhibitors AG490 and Tofacitinib, or Src inhibitor PP2 respectively. Representative phase contrast pictures were taken. ( B) Nuclear fractionation protocol was performed to validate by Western blotting nuclear Fibrillarin enrichment, and enriched cytosolic GAPDH. ( C) Nuclear fractionation was performed from MSC monolayers or from capillary-like structures treated or not with AG490. Total proteins were extracted and representative blots for FOXC2, Fibrillarin, SNAIL, STAT3, and phosphoSTAT3 are presented. Protein data are representative from three distinct experiments. Cropped blots are presented for the sake of clarity and conciseness of data presentation. Uncropped full-length blots are presented in the Supplementary data section, as Supplementary Figure S4B and S4C.

Article Snippet: The polyclonal antibodies against STAT3 (12640 S), Snail (3879 S), FOXC2 (12974 S), and Fibronectin (30903 S), as well as the monoclonal antibody against GAPDH (D4C6R) were all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Generated, Fractionation, Western Blot

Journal: Scientific Reports

Article Title: In vitro biomaterial priming of human mesenchymal stromal/stem cells : implication of the Src/JAK/STAT3 pathway in vasculogenic mimicry

doi: 10.1038/s41598-024-72862-6

Figure Lengend Snippet: STAT3 gene silencing alters MSC capillary-like structure formation , cell chemotaxis , and acquisition of an inflammatory phenotype. ( A) Gene silencing of STAT3 (siSTAT3) was performed in human mesenchymal stromal/stem cells (MSC) as described in the section. Control cells were transfected with a scrambled siRNA sequence (siScrambled). Cell lysates were assessed for GAPDH and STAT3 protein expression. Cropped blots are presented for the sake of clarity and conciseness of data presentation. Uncropped full-length blots are presented in the Supplementary data section, as Supplementary Figure S5A. ( B) Transfected MSC were seeded on top of Cultrex and 3D capillary-like structures generated for 4 h. Representative phase contrast pictures were taken (upper panels) and Wimasis analysis (lower panels) performed, or ( C) real-time cell migration assessed using the xCELLigence as described in the section. ( D) Transfected MSC were seeded as either monolayers (Plastic, white bars) or as capillary-like structures on top of Cultrex (Cultrex, black bars) for 4 h. Total RNA was extracted from triplicate samples for each condition, and gene expression modulation was assessed by RT-qPCR as described in the section. Protein and VM data are representative from two distinct experiments. RT-qPCR data were analyzed from triplicate samples.

Article Snippet: The polyclonal antibodies against STAT3 (12640 S), Snail (3879 S), FOXC2 (12974 S), and Fibronectin (30903 S), as well as the monoclonal antibody against GAPDH (D4C6R) were all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Chemotaxis Assay, Control, Transfection, Sequencing, Expressing, Generated, Migration, Gene Expression, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: In vitro biomaterial priming of human mesenchymal stromal/stem cells : implication of the Src/JAK/STAT3 pathway in vasculogenic mimicry

doi: 10.1038/s41598-024-72862-6

Figure Lengend Snippet: A role for the 37-kDa laminin receptor precursor/67-kDa laminin receptor (RPSA) in MSC 3D capillary-like structure formation. Human mesenchymal stromal/stem cells (MSC) were seeded as either monolayers or as capillary-like structures on top of Cultrex for 4 h. Cell lysates and total RNA were extracted from each condition. (A) Immunoblotting was performed to detect RPSA protein levels. (B) RT-qPCR was performed to assess gene expression in cells cultured as capillary-like structures (Black bars) vs. monolayers (Open bars) upon transient gene silencing of either STAT3 (siSTAT3) or FOXC2 (siFOXC2) as described in the section. (C) Cell lysates were assessed to confirm transient gene silencing of RPSA (siRPSA) at the protein level, and representative capillary-like structures phase-contrast pictures taken in (D) transfected cells (upper panels), or cells treated with a pharmacological RPSA inhibitor (lower panels). Cropped blots are presented for the sake of clarity and conciseness of data presentation. Protein and VM data are representative from two distinct experiments. RT-qPCR data were analyzed from triplicate samples. Uncropped full-length blots are presented in the Supplementary data section, as Supplementary Figure S6A and S6C.

Article Snippet: The polyclonal antibodies against STAT3 (12640 S), Snail (3879 S), FOXC2 (12974 S), and Fibronectin (30903 S), as well as the monoclonal antibody against GAPDH (D4C6R) were all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Quantitative RT-PCR, Gene Expression, Cell Culture, Transfection

Journal: Scientific Reports

Article Title: In vitro biomaterial priming of human mesenchymal stromal/stem cells : implication of the Src/JAK/STAT3 pathway in vasculogenic mimicry

doi: 10.1038/s41598-024-72862-6

Figure Lengend Snippet: Scheme summarizing the STAT3 signaling hub and regulatory impact on capillary-like structure formation , and on the acquisition of an angiogenic , inflammatory , and EMT phenotype. MSC monolayers can recapitulate in vitro VM when cultured on Cultrex. Capillary-like structures can be inhibited in the presence of AG490 and PP2, respectively JAK/STAT3 and Src inhibitors. MSC priming on Cultrex triggers STAT3 expression which, in turn, can regulate the acquisition of an EMT phenotype (increased SNAIL, FOXC2, and RPSA), as well as an angiogenic/inflammatory molecular signature (increased IL6/IL1b/CSF-1, -2). Collectively, this phenotype may form pseudo-vasculature and sustain early pro-angiogenic processes physiologically.

Article Snippet: The polyclonal antibodies against STAT3 (12640 S), Snail (3879 S), FOXC2 (12974 S), and Fibronectin (30903 S), as well as the monoclonal antibody against GAPDH (D4C6R) were all from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, Cell Culture, Expressing