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Millipore stat3 inhibitor sta21

Lysates of platelets stimulated with collagen in the presence of PL were probed for total and phospho-JAK2 (A) and <t>STAT3</t> (B). STAT3 phosphorylation was also probed in platelets treated with a complex of IL-6-sIL-6α either alone or combined with collagen as control (B). Platelet aggregation was also induced by collagen in the presence of a submaximal 50 μM of PL with and without a maximal inhibitory 50 μM of <t>STA21</t> (C, a representative of 6 experiments) or actinomycin D (D, a representative of 6 experiments). Platelets treated with an increasing concentration of the JAK2 inhibitor AG490 were induced to aggregate (E) and to form thrombi on immobilized collagen under flow condition (F, bar = 100 μm, the panel e summaries results from 3 experiments, repeated measure ANOVA, *p < 0.01).

Stat3 Inhibitor Sta21, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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stat3 inhibitor sta21 - by Bioz Stars, 2026-03

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1) Product Images from "Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species †"

Article Title: Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species †

Journal: PLoS ONE

doi: 10.1371/journal.pone.0143964

Lysates of platelets stimulated with collagen in the presence of PL were probed for total and phospho-JAK2 (A) and STAT3 (B). STAT3 phosphorylation was also probed in platelets treated with a complex of IL-6-sIL-6α either alone or combined with collagen as control (B). Platelet aggregation was also induced by collagen in the presence of a submaximal 50 μM of PL with and without a maximal inhibitory 50 μM of STA21 (C, a representative of 6 experiments) or actinomycin D (D, a representative of 6 experiments). Platelets treated with an increasing concentration of the JAK2 inhibitor AG490 were induced to aggregate (E) and to form thrombi on immobilized collagen under flow condition (F, bar = 100 μm, the panel e summaries results from 3 experiments, repeated measure ANOVA, *p < 0.01).

Figure Legend Snippet: Lysates of platelets stimulated with collagen in the presence of PL were probed for total and phospho-JAK2 (A) and STAT3 (B). STAT3 phosphorylation was also probed in platelets treated with a complex of IL-6-sIL-6α either alone or combined with collagen as control (B). Platelet aggregation was also induced by collagen in the presence of a submaximal 50 μM of PL with and without a maximal inhibitory 50 μM of STA21 (C, a representative of 6 experiments) or actinomycin D (D, a representative of 6 experiments). Platelets treated with an increasing concentration of the JAK2 inhibitor AG490 were induced to aggregate (E) and to form thrombi on immobilized collagen under flow condition (F, bar = 100 μm, the panel e summaries results from 3 experiments, repeated measure ANOVA, *p < 0.01).

Techniques Used: Concentration Assay

Image Search Results

Transcriptional factor ICER promotes ADAM9 expression in Th17 cells. (A and B) ICER/CREM-deficient or -sufficient naïve CD4+ T cells cultured under Th17-polarizing conditions for 3 d. (A) Relative ADAM9 mRNA expressions were assessed by qRT-PCR. Cumulative data are shown (mean ± SEM, n = 12). (B) Pro-ADAM9, ADAM9 (active), and β-actin expressions on day 3 were determined by Western blotting. A representative (of six) blot is shown (Left) and cumulative densitometric readings of ADAM9 (active) and β-actin are shown (Right) (mean ± SEM, n = 6). (C) Naïve CD4+ T cells from ICER/CREM-deficient or -sufficient IL-17GFP mice were polarized under Th17 conditions for 3 d. ADAM9 mRNA expression of FACS-sorted GFP+ cells (IL-17A–producing cells) was assessed by qRT-PCR (mean ± SEM, n = 7). (D) Th17-polarized naive CD4+ T cells were cultured in the presence of a STAT3 inhibitor (STA21, 10 μM) or dimethyl sulfoxide (DMSO) for 3 d. Pro-ADAM9, ADAM9 (active), and β-actin expressions were determined by Western blotting. A representative (of five) blot is shown (Left) and cumulative densitometric readings of ADAM9 (active) and β-actin are shown (Right) (mean ± SEM, n = 5). (E) Naive CD4+ T cells from ICER/CREM-deficient IL-17GFP mice were cultured under Th17-polarizing conditions and empty vector (empty) or ADAM9 overexpression (ADAM9 O.E.) plasmids were transfected into cultured T cells on day 1. Representative flow plots on day 3 (Left) and cumulative data (Right) are shown (mean ± SEM, n = 5). (F–H) ICERγ binds to the ADAM9 promoter directly and increases its activity. ICER/CREM-deficient or -sufficient naïve CD4+ T cells were cultured under Th17-polarizing conditions. (F) Schematic representation of the used reporter constructs. Numbers represent the position from the transcription start site of the murine ADAM9 gene. (G) The full-length ADAM9 promoter region (full) or a version containing a mutated CRE binding site (Δ-134/-130) were transfected into Th17-polarized T cells on day 1. Cells were harvested and lysed on day 2. Cumulative results of four independent experiments are shown (mean ± SEM). (H) The FLAG-tagged ICERγ overexpression vector was transfected into ICER/CREM-deficient CD4+ T cells on day one. Cells were harvested and lysed on day 3, and binding of FLAG/ICERγ to the CRE was assessed by ChIP assay. CRE at the intron1 of the ADAM9 gene and CRE at the intron 3 of the adjacent gene (Tm2d2) were used as negative controls for ChIP enrichment. Representative blots from three experiments are shown. *P < 0.05, **P < 0.01.

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-β1

doi: 10.1073/pnas.2023230118

Figure Lengend Snippet: Transcriptional factor ICER promotes ADAM9 expression in Th17 cells. (A and B) ICER/CREM-deficient or -sufficient naïve CD4+ T cells cultured under Th17-polarizing conditions for 3 d. (A) Relative ADAM9 mRNA expressions were assessed by qRT-PCR. Cumulative data are shown (mean ± SEM, n = 12). (B) Pro-ADAM9, ADAM9 (active), and β-actin expressions on day 3 were determined by Western blotting. A representative (of six) blot is shown (Left) and cumulative densitometric readings of ADAM9 (active) and β-actin are shown (Right) (mean ± SEM, n = 6). (C) Naïve CD4+ T cells from ICER/CREM-deficient or -sufficient IL-17GFP mice were polarized under Th17 conditions for 3 d. ADAM9 mRNA expression of FACS-sorted GFP+ cells (IL-17A–producing cells) was assessed by qRT-PCR (mean ± SEM, n = 7). (D) Th17-polarized naive CD4+ T cells were cultured in the presence of a STAT3 inhibitor (STA21, 10 μM) or dimethyl sulfoxide (DMSO) for 3 d. Pro-ADAM9, ADAM9 (active), and β-actin expressions were determined by Western blotting. A representative (of five) blot is shown (Left) and cumulative densitometric readings of ADAM9 (active) and β-actin are shown (Right) (mean ± SEM, n = 5). (E) Naive CD4+ T cells from ICER/CREM-deficient IL-17GFP mice were cultured under Th17-polarizing conditions and empty vector (empty) or ADAM9 overexpression (ADAM9 O.E.) plasmids were transfected into cultured T cells on day 1. Representative flow plots on day 3 (Left) and cumulative data (Right) are shown (mean ± SEM, n = 5). (F–H) ICERγ binds to the ADAM9 promoter directly and increases its activity. ICER/CREM-deficient or -sufficient naïve CD4+ T cells were cultured under Th17-polarizing conditions. (F) Schematic representation of the used reporter constructs. Numbers represent the position from the transcription start site of the murine ADAM9 gene. (G) The full-length ADAM9 promoter region (full) or a version containing a mutated CRE binding site (Δ-134/-130) were transfected into Th17-polarized T cells on day 1. Cells were harvested and lysed on day 2. Cumulative results of four independent experiments are shown (mean ± SEM). (H) The FLAG-tagged ICERγ overexpression vector was transfected into ICER/CREM-deficient CD4+ T cells on day one. Cells were harvested and lysed on day 3, and binding of FLAG/ICERγ to the CRE was assessed by ChIP assay. CRE at the intron1 of the ADAM9 gene and CRE at the intron 3 of the adjacent gene (Tm2d2) were used as negative controls for ChIP enrichment. Representative blots from three experiments are shown. *P < 0.05, **P < 0.01.

Article Snippet: For signal transduction studies, STA21 (STAT3 inhibitor, Santa Cruz Biotechnology) was added to cultures on day 0.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Over Expression, Transfection, Activity Assay, Construct, Binding Assay

Fig. 5 The GPER antagonist G-15 reduces STAT3 nuclear accumulation triggered by estrogens. a Immunofluorescence staining of STAT3 in MDA-MB 231 cells treated for 1 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G-15. Cells were probed with rabbit anti-STAT3 primary antibody followed by FITC-conjugated secondary antibody in order to detect STAT3 displayed by the green signal, whereas the blue signal indicates the nuclei counterstained with DAPI. Images shown are representative of 10 random fields. b Fluorescence intensities of the green signal were quantified in at least 10 random fields in each condition from three independent experiments and data are expressed as fold changes of relative fluorescence units (RFU) upon treatments respect to vehicle-treated cells. Arrows indicate STAT3 nuclear accumulation. (*) indicates p < 0.05

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: Fig. 5 The GPER antagonist G-15 reduces STAT3 nuclear accumulation triggered by estrogens. a Immunofluorescence staining of STAT3 in MDA-MB 231 cells treated for 1 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G-15. Cells were probed with rabbit anti-STAT3 primary antibody followed by FITC-conjugated secondary antibody in order to detect STAT3 displayed by the green signal, whereas the blue signal indicates the nuclei counterstained with DAPI. Images shown are representative of 10 random fields. b Fluorescence intensities of the green signal were quantified in at least 10 random fields in each condition from three independent experiments and data are expressed as fold changes of relative fluorescence units (RFU) upon treatments respect to vehicle-treated cells. Arrows indicate STAT3 nuclear accumulation. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Immunofluorescence, Staining, Fluorescence

Fig. 6 The FAK inhibitor VS-4718 prevents STAT3 nuclear accumulation triggered by estrogens. a Immunofluorescence staining of STAT3 in MDA-MB 231 cells treated for 1 h with 100 nM E2 and 100 nM G1 alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Cells were probed with rabbit anti-STAT3 primary antibody followed by FITC-conjugated secondary antibody in order to detect STAT3 displayed by the green signal, whereas the blue signal indicates the nuclei counterstained with DAPI. Images shown are representative of 10 random fields. b Fluorescence intensities of the green signal were quantified in at least 10 random fields in each condition from three independent experiments and data are expressed as fold changes of relative fluorescence units (RFU) upon treatments respect to vehicle-treated cells. Arrows indicate STAT3 nuclear accumulation. (*) indicates p < 0.05

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: Fig. 6 The FAK inhibitor VS-4718 prevents STAT3 nuclear accumulation triggered by estrogens. a Immunofluorescence staining of STAT3 in MDA-MB 231 cells treated for 1 h with 100 nM E2 and 100 nM G1 alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Cells were probed with rabbit anti-STAT3 primary antibody followed by FITC-conjugated secondary antibody in order to detect STAT3 displayed by the green signal, whereas the blue signal indicates the nuclei counterstained with DAPI. Images shown are representative of 10 random fields. b Fluorescence intensities of the green signal were quantified in at least 10 random fields in each condition from three independent experiments and data are expressed as fold changes of relative fluorescence units (RFU) upon treatments respect to vehicle-treated cells. Arrows indicate STAT3 nuclear accumulation. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Immunofluorescence, Staining, Fluorescence

Fig. 7 c-FOS, EGR1 and CTGF regulation by FAK and STAT3. c-FOS (a), EGR1 (b) and CTGF (c) luciferase promoter activity in MDA-MB 231 cells treated for 18 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G15 or 20 μM STAT3 inhibitor STA21. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction upon which the activities induced by treatments were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. d c-FOS, EGR1 and CTGF mRNA expression measured by real time-PCR in MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G15 or 20 μM STAT3 inhibitor STA21. Values normalized to the 18 s expression are shown as fold changes of the mRNA expression induced by treatments compared to cells treated with vehicle (−). e-f Immunoblots showing c-FOS, EGR1 and CTGF protein expression in MDA-MB 231 cells treated for 4 h with 100 nM E2 (e) and 100 nM G1 (f) alone or in combination with 20 μM STAT3 inhibitor STA21. Side panels show densitometric analysis of the immunoblots normalized to β-actin. g c-FOS, EGR1 and CTGF mRNA expression measured by real time-PCR in MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Values normalized to the 18 s expression are shown as fold changes of the mRNA expression induced by treatments compared to cells treated with vehicle (−). Immunoblots showing c-FOS, EGR1 and CTGF protein expression in MDA-MB 231 cells treated for 4 h with 100 nM E2 (h) and 100 nM G1 (i) alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Side panels show densitometric analysis of the immunoblots normalized to β-actin. Results shown are representative of three independent experiments. (*) indicates p < 0.05

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: Fig. 7 c-FOS, EGR1 and CTGF regulation by FAK and STAT3. c-FOS (a), EGR1 (b) and CTGF (c) luciferase promoter activity in MDA-MB 231 cells treated for 18 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G15 or 20 μM STAT3 inhibitor STA21. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction upon which the activities induced by treatments were calculated. Each data point represents the mean ± SD of three independent experiments performed in triplicate. d c-FOS, EGR1 and CTGF mRNA expression measured by real time-PCR in MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 100 nM GPER antagonist G15 or 20 μM STAT3 inhibitor STA21. Values normalized to the 18 s expression are shown as fold changes of the mRNA expression induced by treatments compared to cells treated with vehicle (−). e-f Immunoblots showing c-FOS, EGR1 and CTGF protein expression in MDA-MB 231 cells treated for 4 h with 100 nM E2 (e) and 100 nM G1 (f) alone or in combination with 20 μM STAT3 inhibitor STA21. Side panels show densitometric analysis of the immunoblots normalized to β-actin. g c-FOS, EGR1 and CTGF mRNA expression measured by real time-PCR in MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Values normalized to the 18 s expression are shown as fold changes of the mRNA expression induced by treatments compared to cells treated with vehicle (−). Immunoblots showing c-FOS, EGR1 and CTGF protein expression in MDA-MB 231 cells treated for 4 h with 100 nM E2 (h) and 100 nM G1 (i) alone or in combination with 1 μM FAK kinase inhibitor VS-4718. Side panels show densitometric analysis of the immunoblots normalized to β-actin. Results shown are representative of three independent experiments. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

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

Fig. 9 The STAT3 inhibitor STA21 suppresses the migration of TNBC cells induced by E2 and G1. a Boyden Chamber assays showing the migration of MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 20 μM STAT3 inhibitor STA21. The results are shown as cells migrating through the membrane at the bottom of the well upon treatments respect to vehicle (−). Results shown are representative of three independent experiments. b Cell migration was evaluated by wound-healing assay in MDA-MB 231 cells treated for 24 h with 100 nM E2 and 100 nM G1 alone or in combination with 20 μM STAT3 inhibitor STA21. White dotted lines indicate the wound borders at the beginning of the assay and recorded 24 h post- scratching. Results shown are representative of three independent experiments. (*) indicates p < 0.05

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Focal adhesion kinase (FAK) activation by estrogens involves GPER in triple-negative breast cancer cells.

doi: 10.1186/s13046-019-1056-8

Figure Lengend Snippet: Fig. 9 The STAT3 inhibitor STA21 suppresses the migration of TNBC cells induced by E2 and G1. a Boyden Chamber assays showing the migration of MDA-MB 231 cells treated for 4 h with 100 nM E2 and 100 nM G1 alone or in combination with 20 μM STAT3 inhibitor STA21. The results are shown as cells migrating through the membrane at the bottom of the well upon treatments respect to vehicle (−). Results shown are representative of three independent experiments. b Cell migration was evaluated by wound-healing assay in MDA-MB 231 cells treated for 24 h with 100 nM E2 and 100 nM G1 alone or in combination with 20 μM STAT3 inhibitor STA21. White dotted lines indicate the wound borders at the beginning of the assay and recorded 24 h post- scratching. Results shown are representative of three independent experiments. (*) indicates p < 0.05

Article Snippet: STAT3 transcription factor signaling inhibitor STA21 and Focal Adhesion Kinase selective inhibitor VS-4718 were bought from Santa Cruz Biotechnology (DBA, Milan, Italy).

Techniques: Migration, Membrane, Wound Healing Assay

A: IL-6 levels in conditioned media from mock-infected control (cont.) and VR1814-infected amniotic epithelial cells (AmEpCs) quantified by enzyme-linked immunosorbent assay. Similar results were obtained with four cell preparations. B: Human cytomegalovirus (HCMV) infection activates STAT3 and up-regulates anti-apoptotic proteins. Cell lysates from mock-infected control and VR1814-infected AmEpCs or ARPE-19 cells at indicated times were immunoblotted with antibodies to phospho-STAT3 (pSTAT3), STAT3, Bcl-2, Bcl-xL, survivin, and actin (loading control). Results are representative of at least three independent experiments. C and D: Immunostaining of survivin (B), Bcl-xL (C), and immediate early (IE) 1 (CH443) in mock-infected control and VR1814-infected cells. Nuclei were stained with DAPI. Blue dashes delineate foci of infection. E: Effects of STAT3 inhibitors on Bcl-xL and survivin expression. Mock-infected control and VR1814-infected AmEpCs were cultured with medium alone (no treat), STAT3 inhibitors S31-201 (100 μmol/L), WP6001 (5 μmol/L), or STA21 (50 μmol/L), or the vehicle dimethyl sulfoxide (DMSO). Cell lysates at 3 days postinfection (dpi) were immunoblotted with antibodies to Bcl-xL, survivin, and actin (loading control). Results are representative of at least four independent experiments. F: Cell lysates from mock-infected control and VR1814-infected AmEpCs from different gestational ages were immunoblotted with antibodies to Bcl-2, Bcl-xL, survivin, and actin (loading control). Scale bar = 50 μm (C and D). Original magnification, ×200 (C and D).

Journal: The American Journal of Pathology

Article Title: Persistent Cytomegalovirus Infection in Amniotic Membranes of the Human Placenta

doi: 10.1016/j.ajpath.2016.07.016

Figure Lengend Snippet: A: IL-6 levels in conditioned media from mock-infected control (cont.) and VR1814-infected amniotic epithelial cells (AmEpCs) quantified by enzyme-linked immunosorbent assay. Similar results were obtained with four cell preparations. B: Human cytomegalovirus (HCMV) infection activates STAT3 and up-regulates anti-apoptotic proteins. Cell lysates from mock-infected control and VR1814-infected AmEpCs or ARPE-19 cells at indicated times were immunoblotted with antibodies to phospho-STAT3 (pSTAT3), STAT3, Bcl-2, Bcl-xL, survivin, and actin (loading control). Results are representative of at least three independent experiments. C and D: Immunostaining of survivin (B), Bcl-xL (C), and immediate early (IE) 1 (CH443) in mock-infected control and VR1814-infected cells. Nuclei were stained with DAPI. Blue dashes delineate foci of infection. E: Effects of STAT3 inhibitors on Bcl-xL and survivin expression. Mock-infected control and VR1814-infected AmEpCs were cultured with medium alone (no treat), STAT3 inhibitors S31-201 (100 μmol/L), WP6001 (5 μmol/L), or STA21 (50 μmol/L), or the vehicle dimethyl sulfoxide (DMSO). Cell lysates at 3 days postinfection (dpi) were immunoblotted with antibodies to Bcl-xL, survivin, and actin (loading control). Results are representative of at least four independent experiments. F: Cell lysates from mock-infected control and VR1814-infected AmEpCs from different gestational ages were immunoblotted with antibodies to Bcl-2, Bcl-xL, survivin, and actin (loading control). Scale bar = 50 μm (C and D). Original magnification, ×200 (C and D).

Article Snippet: The STAT3 inhibitors S31-201, WP1006, and STA21 were purchased from Santa Cruz Biotechnology.

Techniques: Infection, Control, Enzyme-linked Immunosorbent Assay, Immunostaining, Staining, Expressing, Cell Culture

Journal: PLoS ONE

Article Title: Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species †

doi: 10.1371/journal.pone.0143964

Figure Lengend Snippet: Lysates of platelets stimulated with collagen in the presence of PL were probed for total and phospho-JAK2 (A) and STAT3 (B). STAT3 phosphorylation was also probed in platelets treated with a complex of IL-6-sIL-6α either alone or combined with collagen as control (B). Platelet aggregation was also induced by collagen in the presence of a submaximal 50 μM of PL with and without a maximal inhibitory 50 μM of STA21 (C, a representative of 6 experiments) or actinomycin D (D, a representative of 6 experiments). Platelets treated with an increasing concentration of the JAK2 inhibitor AG490 were induced to aggregate (E) and to form thrombi on immobilized collagen under flow condition (F, bar = 100 μm, the panel e summaries results from 3 experiments, repeated measure ANOVA, *p < 0.01).

Article Snippet: Commercial reagents used in the study included: PL (Cayman Chemical Co., Ann Arbor, MI), the STAT3 inhibitor STA21 (Sigma Aldrich, St. Louis, MO), the JAK2-inhbitor AG490 (InvivoGen, San Diego, CA), the Syk inhibitor SykII (Merck Millipore, Billerica, MA), human recombinant IL-6 (R & D Systems, Minneapolis, MN), the extracellular domain of human IL-6 receptor-α (R & D Systems), Actinomycin (Sigma Aldrich), apocynin (Abcam Biochemicals.

Techniques: Concentration Assay

Effects of STA21 on platelet aggregation: PRP was incubated with STA21 or DMSO vehicle control (0.1%) for 10 min at 37°C and then induced to aggregate with various concentrations of collagen (A, Univariate Repeated Measures for each collagen dose compared to baseline, n = 9, *p < 0.001) or CRP (B, compared to baseline, n = 3, *p < 0.001). Secretion from α-granule was measured by detecting surface expression of CD62P by flow cytometry after platelets were stimulated with 2 μg/ml of collagen for 10 min (C, Univariate Repeated Measures for each CRP dose compared to baseline, n = 6, *p < 0.001). ATP (D, n = 6) and serotonin (E, n = 3) released from dense granules were measured by whole blood aggregometry and ELISA, respectively, in the supernatant of platelets stimulated with 2 μg/ml of collagen. The data for the panels A-C are also presented as box blots in Supplemental Figure 5. Thrombus formation in vitro was induced by perfusing STA21-treated (20 μm, 10 min at 37°C) blood over immobilized collagen for 1 min at a flow rate of 1 ml/min. Representative images show platelet thrombi in the presence (F) and absence (G) of STA21 (Bar = 200 μm). The areas covered by thrombi were quantified in 6 random images from each experiment (H, n = 3 separate sets of experiments, *p < 0.001). For mouse assay, C57BL/6J mice were daily injected with 4 or 8 mg/kg body weight of STA21 or vehicle control through tail veins for 3 days and collagen-induced platelet aggregation was measured on the third day on an optical aggregomater (I, n = 8, *p < 0.01).

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: Effects of STA21 on platelet aggregation: PRP was incubated with STA21 or DMSO vehicle control (0.1%) for 10 min at 37°C and then induced to aggregate with various concentrations of collagen (A, Univariate Repeated Measures for each collagen dose compared to baseline, n = 9, *p < 0.001) or CRP (B, compared to baseline, n = 3, *p < 0.001). Secretion from α-granule was measured by detecting surface expression of CD62P by flow cytometry after platelets were stimulated with 2 μg/ml of collagen for 10 min (C, Univariate Repeated Measures for each CRP dose compared to baseline, n = 6, *p < 0.001). ATP (D, n = 6) and serotonin (E, n = 3) released from dense granules were measured by whole blood aggregometry and ELISA, respectively, in the supernatant of platelets stimulated with 2 μg/ml of collagen. The data for the panels A-C are also presented as box blots in Supplemental Figure 5. Thrombus formation in vitro was induced by perfusing STA21-treated (20 μm, 10 min at 37°C) blood over immobilized collagen for 1 min at a flow rate of 1 ml/min. Representative images show platelet thrombi in the presence (F) and absence (G) of STA21 (Bar = 200 μm). The areas covered by thrombi were quantified in 6 random images from each experiment (H, n = 3 separate sets of experiments, *p < 0.001). For mouse assay, C57BL/6J mice were daily injected with 4 or 8 mg/kg body weight of STA21 or vehicle control through tail veins for 3 days and collagen-induced platelet aggregation was measured on the third day on an optical aggregomater (I, n = 8, *p < 0.01).

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Mouse Assay, Injection

Platelet function of STAT3Δ/Δ mice: Platelets from pSTAT3Δ/Δ and STAT3F/F littermates were induced to aggregate by 0.5, 0.75, or 5 μg/ml of collagen (A-C) and data from 32 mice/group were quantified (D, *p < 0.01). CD62p expression was measured after platelets were stimulated with 0.5, 0.75 or 5 μg/ml of collagen. The comparison was made between WT and STAT3 KO platelets at each collagen level with a two-way ANOVA (E, n = 12, *p < 0.001). No interaction between litter and genotype was found at any of these collagen doses. Calcium influx was detected in platelets stimulated with 0.75 μg/ml of collagen (F, *p < 0.003). Blood was perfused over immobilized collagen for 10 min at a flow rate of 1 ml/min to measure thrombus formation (G, representative images, bar = 50 μm), which was quantified by measuring surface areas covered by platelet thrombi (H, n = 10, *p < 0.001).

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: Platelet function of STAT3Δ/Δ mice: Platelets from pSTAT3Δ/Δ and STAT3F/F littermates were induced to aggregate by 0.5, 0.75, or 5 μg/ml of collagen (A-C) and data from 32 mice/group were quantified (D, *p < 0.01). CD62p expression was measured after platelets were stimulated with 0.5, 0.75 or 5 μg/ml of collagen. The comparison was made between WT and STAT3 KO platelets at each collagen level with a two-way ANOVA (E, n = 12, *p < 0.001). No interaction between litter and genotype was found at any of these collagen doses. Calcium influx was detected in platelets stimulated with 0.75 μg/ml of collagen (F, *p < 0.003). Blood was perfused over immobilized collagen for 10 min at a flow rate of 1 ml/min to measure thrombus formation (G, representative images, bar = 50 μm), which was quantified by measuring surface areas covered by platelet thrombi (H, n = 10, *p < 0.001).

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Expressing

Hematological measurements of pSTAT3 Δ/Δ and STAT3 F/F mice *

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: Hematological measurements of pSTAT3 Δ/Δ and STAT3 F/F mice *

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Cell Counting

STAT3 phosphorylation in human platelets: Washed platelets were incubated with various concentrations of collagen (A) or CRP (B) for 10 min at 37°C. Platelet lysates were probed with antibodies against Tyr705 phosphorylated, Ser727 phosphorylated, and total STAT3. Aliquots of platelets were collected and probed for STAT3 phosphorylation over a 15 min after platelets were stimulated with 5 μg/ml of collagen. The optical density of immunoreactive bands of STAT3 phosphorylation was recorded (C). STAT3 phosphorylation induced by 5 μg/ml of collagen was measured in the presence of STA21 (D). STAT3 phosphorylation was also determined in TRAP-treated platelets (E). STAT1 and STAT5 phosphorylation was probed in collagen-stimulated platelet lysates (F). Human washed platelets were first treated with one of two Syk inhibitors for 10 min and then stimulated with 5 μg/ml of collagen. Platelet lysates were probed for the phosphorylation of STAT3 (G) and PLCγ2 (H). STA21-treated platelets were stimulated with collagen and probed for Syk phosphorylation (I). Phosphorylated and total PLCγ2 was probed in platelets treated with various doses of collagen in the presence of increasing doses of STA21 (J). Panel figures represent 3–7 separate experiments.

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: STAT3 phosphorylation in human platelets: Washed platelets were incubated with various concentrations of collagen (A) or CRP (B) for 10 min at 37°C. Platelet lysates were probed with antibodies against Tyr705 phosphorylated, Ser727 phosphorylated, and total STAT3. Aliquots of platelets were collected and probed for STAT3 phosphorylation over a 15 min after platelets were stimulated with 5 μg/ml of collagen. The optical density of immunoreactive bands of STAT3 phosphorylation was recorded (C). STAT3 phosphorylation induced by 5 μg/ml of collagen was measured in the presence of STA21 (D). STAT3 phosphorylation was also determined in TRAP-treated platelets (E). STAT1 and STAT5 phosphorylation was probed in collagen-stimulated platelet lysates (F). Human washed platelets were first treated with one of two Syk inhibitors for 10 min and then stimulated with 5 μg/ml of collagen. Platelet lysates were probed for the phosphorylation of STAT3 (G) and PLCγ2 (H). STA21-treated platelets were stimulated with collagen and probed for Syk phosphorylation (I). Phosphorylated and total PLCγ2 was probed in platelets treated with various doses of collagen in the presence of increasing doses of STA21 (J). Panel figures represent 3–7 separate experiments.

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Incubation

Co-immunoprecipitation: Washed human platelets were stimulated with 5 μg/ml of collagen and platelet lysates were incubated with a STAT3 antibody followed by immunoprecipitation (IP) with protein A sepharose beads. Precipitated proteins were probed with antibodies to STAT3, Syk, PLCγ2, and actin (A, isotype IgG as control and STAT3 as loading control, platelet lysate [PL] as positive control). The same technique was used to immunoprecipitate PLCγ2 and probe for PLCγ2, Syk, STAT3, and actin (B). STAT3 and PLCγ2 were also immunoprecipitated with antibodies specifically against phosphorylated PLCγ2 and phosphorylated STAT3, respectively (C). STAT3 was co-immunoprecipitated with a PLCγ2 antibody from lysates of platelets stimulated with 5 μg/ml of collagen in the presence of increasing doses of STA21 (D). PLCγ2 phosphorylation was measured in platelets from pSTAT3Δ/Δ and STAT3F/F mice stimulated with increasing doses of collagen (E). Panels in the figure represent of 3–6 separate experiments.

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: Co-immunoprecipitation: Washed human platelets were stimulated with 5 μg/ml of collagen and platelet lysates were incubated with a STAT3 antibody followed by immunoprecipitation (IP) with protein A sepharose beads. Precipitated proteins were probed with antibodies to STAT3, Syk, PLCγ2, and actin (A, isotype IgG as control and STAT3 as loading control, platelet lysate [PL] as positive control). The same technique was used to immunoprecipitate PLCγ2 and probe for PLCγ2, Syk, STAT3, and actin (B). STAT3 and PLCγ2 were also immunoprecipitated with antibodies specifically against phosphorylated PLCγ2 and phosphorylated STAT3, respectively (C). STAT3 was co-immunoprecipitated with a PLCγ2 antibody from lysates of platelets stimulated with 5 μg/ml of collagen in the presence of increasing doses of STA21 (D). PLCγ2 phosphorylation was measured in platelets from pSTAT3Δ/Δ and STAT3F/F mice stimulated with increasing doses of collagen (E). Panels in the figure represent of 3–6 separate experiments.

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Immunoprecipitation, Incubation, Positive Control

Effect of IL-6 on platelet and HEK293 cells. STAT3 phosphorylation was measured in platelets stimulated with IL-6/sIL-6R or IL-6 in the absence (A) and presence (B) of increasing concentrations of collagen. HEK293 cells transiently expressing human Syk and PLCγ2 were stimulated with 10 ng/ml of IL-6 for 1 hr at 37°C and then lysed. Cell lysates were probed for total and phosphorylated STAT3 (C). These cells were also stimulated with IL-6 in the presence of STA21 and probed for STAT3 phosphorylation (D). Resting and IL-6 stimulated HEK293 cells were lysed and incubated with a STAT3 antibody followed by immunoprecipitation by protein A coupled beads (E). Immunoprecipitated proteins were probed for Syk, PLCγ2, and STAT3. Non-immune isotype IgG was used as negative control. The figure represent of 3–4 separate experiments.

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: Effect of IL-6 on platelet and HEK293 cells. STAT3 phosphorylation was measured in platelets stimulated with IL-6/sIL-6R or IL-6 in the absence (A) and presence (B) of increasing concentrations of collagen. HEK293 cells transiently expressing human Syk and PLCγ2 were stimulated with 10 ng/ml of IL-6 for 1 hr at 37°C and then lysed. Cell lysates were probed for total and phosphorylated STAT3 (C). These cells were also stimulated with IL-6 in the presence of STA21 and probed for STAT3 phosphorylation (D). Resting and IL-6 stimulated HEK293 cells were lysed and incubated with a STAT3 antibody followed by immunoprecipitation by protein A coupled beads (E). Immunoprecipitated proteins were probed for Syk, PLCγ2, and STAT3. Non-immune isotype IgG was used as negative control. The figure represent of 3–4 separate experiments.

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Expressing, Incubation, Immunoprecipitation, Negative Control

A schematic crosstalk between IL-6 and collagen signal pathways: Data from this study support a model of crosstalk between collagen-induced and cytokine-mediated STAT3 signals in platelets. This crosstalk may be active during inflammation where the secretion of the proinflammatory cytokine IL-6 and the membrane shedding of IL-6 receptor (gp80) result in the formation of soluble IL-6•sIL-6R complex that binds to gp130 on platelets to activate STAT3. The activated STAT3 serves as a protein scaffold to bring the kinase Syk to the vicinity of the substrate PLCγ2 to enhance or accelerate PLCγ2 phosphorylation in response to collagen stimulation. Activated PLCγ2 could then hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-triphosphate (IP3) to mobilize calcium.

Journal: Circulation

Article Title: STAT3 Regulates Collagen-Induced Platelet Aggregation Independent of its Transcription Factor Activity

doi: 10.1161/CIRCULATIONAHA.112.132126

Figure Lengend Snippet: A schematic crosstalk between IL-6 and collagen signal pathways: Data from this study support a model of crosstalk between collagen-induced and cytokine-mediated STAT3 signals in platelets. This crosstalk may be active during inflammation where the secretion of the proinflammatory cytokine IL-6 and the membrane shedding of IL-6 receptor (gp80) result in the formation of soluble IL-6•sIL-6R complex that binds to gp130 on platelets to activate STAT3. The activated STAT3 serves as a protein scaffold to bring the kinase Syk to the vicinity of the substrate PLCγ2 to enhance or accelerate PLCγ2 phosphorylation in response to collagen stimulation. Activated PLCγ2 could then hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-triphosphate (IP3) to mobilize calcium.

Article Snippet: The STAT3 inhibitor STA21 [(S)-Ochromycinone Deoxytetrangomycin (C19H14O4)] 16 was purchased from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques:

Review

Structured Review

Images

1) Product Images from "Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species †"

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