Journal: Neural regeneration research

Article Title: Microglial depletion impairs glial scar formation and aggravates inflammation partly by inhibiting STAT3 phosphorylation in astrocytes after spinal cord injury.

doi: 10.4103/1673-5374.357912

Figure Lengend Snippet: Figure 5 ｜LPS-activated microglia promote astrocyte proliferation in vitro. (A) EdU staining and cellular localization with astrocytes cocultured under different conditions. (B) Western blotting analysis of STAT3, pSTAT3 and GFAP in the mixed cells after 24 hours of incubation. (C, D) The quantification of pSTAT3 (C, n = 3) and GFAP (D, n = 3) was normalized to β-actin. (E) LPS-activated microglia significantly increases the proportion of EdU+/GFAP+ astrocytes, which was reversed by STA21 (an inhibitor of STAT3) pretreatment (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). Data are expressed as the mean ± SD. Western blot experiments were repeated three times. Immunofluorescence staining was repeated six times. DAPI: 4′,6-Diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; GFAP: glial fibrillary acidic protein; LPS: lipopolysaccharide; pSTAT3: phosphorylated STAT3; SCI: spinal cord injury; STAT3: signal transducers and activators of transcription 3.

Article Snippet: To assess the role of STAT3 signaling, astrocytes were pretreated with 10 μM STA21 (an inhibitor of STAT3, Selleck, Shanghai, China, Cat# S7951) for 72 hours before coculture.

Techniques: In Vitro, Staining, Western Blot, Incubation, Immunofluorescence

Journal: Cell Death Discovery

Article Title: Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

doi: 10.1038/s41420-020-0272-5

Figure Lengend Snippet: a Levels of Tyr705-phosphorylated STAT3 (p-STAT3) and total STAT3 in LNCaP cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 as shown by western blotting. Cells were pre-treated with IL-6 (0.1, 1 or 10 ng/mL) for 16 h ( N = 5). b Levels of Tyr705-phosphorylated STAT3 (p-STAT3) in 22Rv1 cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 as shown by western blotting. Cells were pre-treated with 10 ng/mL IL-6 for 16 h ( N = 4). STAT3 phosphorylation at Tyr705 indicates STAT3 activation upon IL-6 treatment. The blots in a , b were cut into two pieces and probed with antibodies against p-STAT3 and GAPDH separately. The p-STAT3 blots were then stripped and blotted with an antibody against STAT3. c Survival of LNCaP cells as assessed by the WST assay after treatment with 1 μM doxorubicin for 48 h in the presence or absence of IL-6 and 1 μM JAK kinase inhibitor Ruxolitinib or 20 μM STAT3 inhibitor STA-21. Cells were pre-treated with IL-6 (0.1, 1 or 10 ng/mL) for 16 h. Cell survival is shown as fold change relative to cell survival in the absence of JAK/STAT3 inhibitor (S.E.M. is indicated by bars; N = 6). d Left panel: p53 levels in LNCaP cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 and 1 μM JAK kinase inhibitor Ruxolitinib or 20 μM STAT3 inhibitor STA-21. Cells were pre-treated with IL-6 (10 ng/mL) for 16 h. The blot was cut into two pieces and probed with anti-p53 DO-1 and anti-GAPDH antibody separately. Samples to be compared were loaded on the same gel and transferred onto the same membrane. Right panel: quantification of the western blot data shown as ratio of p53 to GAPDH (S.E.M. is indicated by bars; N = 2). e STAT3 protein phosphorylation and total STAT3 protein levels in LNCaP cells after treatment with 1 μM doxorubicin for 8 h in the presence or absence of IL-6 and 1 μM JAK kinase inhibitor Ruxolitinib or 20 μM STAT3 inhibitor STA-21. Cells were pre-treated with 10 ng/mL IL-6 for 16 h ( N = 2). The blot was cut into two pieces and probed with antibodies against p-STAT3 and GAPDH separately. The p-STAT3 blot was then stripped and blotted with anti-STAT3 antibody.

Article Snippet: The inhibitors used were JAK inhibitors Ruxolitinib (INCB018424 Selleckchem, Rungsted, Denmark) and Pyridone 6 (Calbiochem) and STAT3 inhibitors STA-21 and Stattic (both from Selleckchem, Denmark).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, WST Assay, Membrane

Journal: Cell Death Discovery

Article Title: Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

doi: 10.1038/s41420-020-0272-5

Figure Lengend Snippet: a Oncoprint from cBioportal of two selected prostate cancer studies showing genetic alteration profiles of IL-6R, STAT3, MDM2, and TP53. Each row represents the genetic alteration according to the figure legend with individual patient in the column. b mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) of IL-6R, STAT3 or MDM2 from prostate adenocarcinoma (prad) of the TCGA PanCancer Atlas study based on patients that have no TP53 alterations or putative driver TP53 mutations (missense or truncating). N = 414 no alterations, and N = 56 putative driver mutations. Mann–Whitney test, for IL-6R ** p = 0.003, STAT3 p = 0.47 and MDM2 *** p < 0.0001. c Scatter plots of mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) of IL-6R, JAK1, JAK2, STAT3 vs MDM2 in prostate adenocarcinoma patients of the TCGA PanCancer Atlas study that have no alterations in TP53. Indicated r and p values are analysis by Spearman correlation. N = 414 in all plots. d Scatter plots of mRNA expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) of IL-6R, JAK1, JAK2, STAT3 vs MDM2 in prostate adenocarcinoma patients of the TCGA PanCancer Atlas study that have putative driver alterations (missense and truncating) in TP53. Indicated r and p values are analysis by Spearman correlation. N = 56 in all plots, except the correlation with JAK2 N = 55. e Survival of patients with metastatic prostate adenocarcinoma from the study by Abida et al. . N is indicated in the figure. Black dot indicates censored patient. Left panel; patients with no TP53 alterations were grouped as having IL-6R alterations (amplification ( N = 12), mRNA high ( N = 1) missense mutation ( N = 1)) or having no IL-6R alterations. Log-rank (Mantel–Cox) test p = 0.09 or Gehan–Breslow–Wilcoxon test ** p = 0.01. Right panel; patients with putative driver TP53 mutations (missense, truncating, inframe) were grouped as having IL-6R alterations (amplification ( N = 4), mRNA high ( N = 3)) or having no IL-6R alterations. Log-rank (Mantel–Cox) test p = 0.5 or Gehan–Breslow–Wilcoxon test p = 0.9.

Article Snippet: The inhibitors used were JAK inhibitors Ruxolitinib (INCB018424 Selleckchem, Rungsted, Denmark) and Pyridone 6 (Calbiochem) and STAT3 inhibitors STA-21 and Stattic (both from Selleckchem, Denmark).

Techniques: Expressing, MANN-WHITNEY, Amplification, Mutagenesis

Journal: Cell Death Discovery

Article Title: Interleukin-6 derived from cancer-associated fibroblasts attenuates the p53 response to doxorubicin in prostate cancer cells

doi: 10.1038/s41420-020-0272-5

Figure Lengend Snippet: CAFs secrete IL-6, which binds to IL-6 receptors (IL-6R) expressed on cancer cells. IL-6 binding to its receptor leads to activation of JAK kinase and STAT3, respectively. STAT3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding. This stimulates Mdm2-mediated p53 ubiquitination and p53 degradation in the proteasome, thereby inhibiting upregulation of the p53 target Bax upon treatment with doxorubicin. As a result, prostate cancer cell survival is enhanced.

Article Snippet: The inhibitors used were JAK inhibitors Ruxolitinib (INCB018424 Selleckchem, Rungsted, Denmark) and Pyridone 6 (Calbiochem) and STAT3 inhibitors STA-21 and Stattic (both from Selleckchem, Denmark).

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Translocation Assay, Ubiquitin Proteomics

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

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: 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

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: 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

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: 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

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: 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

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

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

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

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

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

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

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:

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

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: Primer sets used for PCR and targeted sequences for knockdown studies

Article Snippet: Native or HCK promoter-driven luciferase reporter transduced BCWM.1 or TMD8 cells were treated with inhibitors to transcription factors (TFs) STAT3 (STA-21; Selleck Chemicals, Houston, TX; Galiellalactone, Tocris Bioscience, Minneapolis, MN); AP1 (SP100030; SR 11302; Tocris Bioscience), and NF-κB (ACHP; Tocris Bioscience; QNZ; Triptolide [PG490]; Selleck Chemicals) for HCK transcription or promoter activity studies.

Techniques: Clone Assay

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: TF expression, activation, and the impact of PAX5 on HCK transcription in MYD88-mutated lymphoma cells. (A) Western blot studies depicting protein expression levels of PAX5, STAT3, NF-kB (NF-κB-p65), and AP-1 complex members (JunB, c-Jun, JunD) predicted by TF promoter-binding assay and PROMO analysis as HCK promoter binding TFs in MYD88-mutated WM and ABC-DLBCL cell lines (BCWM.1, MWCL-1, TMD-8, HBL-1, OCI-Ly3, and SU-DHL-2) and MYD88 wild-type B-cell lymphoma (OCI-Ly7, OCI-Ly19, Ramos) and myeloma cells (RPMI-8226, MM.1S). The HCK protein expression levels and the phosphorylation levels of mutated MYD88–directed TFs STAT3, NF-κB-p65, and AP-1 complex members (JunB, c-Jun) were also detected. GAPDH protein expression was used to demonstrate uniform protein loading. (B) The regulation of HCK transcription by PAX5 was assessed by lentiviral knockdown of PAX5 with 2 distinct shRNAs in MYD88-mutated BCWM.1 and TMD-8 cells and compared with scrambled control vector. Quantitative RT-PCR was performed after day 5 of lentiviral transduction. HCK protein levels and knockdown efficiencies for PAX5 were analyzed by western blot at the same time as the sample collection for HCK mRNA quantification. GAPDH was used for loading control. (C) The regulation of PAX5 by mutated MYD88 was assessed by lentiviral-mediated knockdown of MYD88 in MYD88-mutated BCWM.1 and TMD-8 cells using 2 distinct shRNAs and compared with scrambled control vector. Protein levels of PAX5 are shown, and GADPH served as a protein loading control. (D) Transcriptome analysis depicting PAX5 transcript levels in CD19-selected bone marrow LPCs from MYD88-mutated WM patients, and MYD88 wild-type WM patients; peripheral CD19-selected B cells and CD19- and CD27-selected memory B cells from healthy donors; and CD138-selected bone marrow plasma cells from healthy donors. ***P < .001.

Article Snippet: Native or HCK promoter-driven luciferase reporter transduced BCWM.1 or TMD8 cells were treated with inhibitors to transcription factors (TFs) STAT3 (STA-21; Selleck Chemicals, Houston, TX; Galiellalactone, Tocris Bioscience, Minneapolis, MN); AP1 (SP100030; SR 11302; Tocris Bioscience), and NF-κB (ACHP; Tocris Bioscience; QNZ; Triptolide [PG490]; Selleck Chemicals) for HCK transcription or promoter activity studies.

Techniques: Expressing, Activation Assay, Western Blot, Binding Assay, Plasmid Preparation, Quantitative RT-PCR, Transduction

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: ChIP studies assessing STAT3, NF-kB, and AP-1 TF binding to the HCK promoter. The fold enrichments of HCK promoter-specific sequence assessed by quantitative PCR following ChIP with ChIP grade antibodies to STAT3, NF-kB-p65, JunB, and c-Jun in MYD88-mutated WM (BCWM.1, MWCL-1) and ABC-DLBCL (TMD-8, HBL-1, OCI-Ly3) cells, and MYD88 wild-type lymphoma cells (OCI-Ly7, OCI-Ly19). Antibody to GAPDH was used as control antibody.

Article Snippet: Native or HCK promoter-driven luciferase reporter transduced BCWM.1 or TMD8 cells were treated with inhibitors to transcription factors (TFs) STAT3 (STA-21; Selleck Chemicals, Houston, TX; Galiellalactone, Tocris Bioscience, Minneapolis, MN); AP1 (SP100030; SR 11302; Tocris Bioscience), and NF-κB (ACHP; Tocris Bioscience; QNZ; Triptolide [PG490]; Selleck Chemicals) for HCK transcription or promoter activity studies.

Techniques: Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, ChIP-chip

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: Deletion of STAT3, AP-1, and NF-kB consensus DNA motifs reduces HCK promoter activity and their binding to HCK promoter in MYD88-mutated WM and ABC-DLBCL cells. HCK promoter activity was assessed following deletion of AP-1, STAT3, and NF-kB binding sites in TNF-α–stimulated HEK-293 cells (A), and MYD88-mutated BCWM.1 and TMD-8 cells (B). (C) The binding of biotin-labeled HCK promoter to the corresponding TFs NF-kB, STAT3, and AP-1 was also reduced by indicated mutants in pull-down assays. **P < .01; ***P < .001.

Article Snippet: Native or HCK promoter-driven luciferase reporter transduced BCWM.1 or TMD8 cells were treated with inhibitors to transcription factors (TFs) STAT3 (STA-21; Selleck Chemicals, Houston, TX; Galiellalactone, Tocris Bioscience, Minneapolis, MN); AP1 (SP100030; SR 11302; Tocris Bioscience), and NF-κB (ACHP; Tocris Bioscience; QNZ; Triptolide [PG490]; Selleck Chemicals) for HCK transcription or promoter activity studies.

Techniques: Activity Assay, Binding Assay, Labeling

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: Inhibitors to STAT3, NF-κB, and AP-1 reduce HCK transcription in MYD88-mutated WM and ABC-DLBCL cells. (A) HCK promoter activities indicated by RLU% were determined by HCK promoter-driven luciferase reporter assay following treatment by inhibitors to STAT3 (STA-21), NF-kB (QNZ), and AP-1 (SR 11302) at one-fifth to one-twenty-fifth of their 50% effective concentration (EC50) levels for 6 hours in BCWM.1 WM and TMD-8 ABC-DLBCL cells. (B) HCK mRNA levels were determined by TaqMan Gene Expression Assay following treatment by inhibitors to STAT3 (Galiellalactone), NF-kB (ACHP), and AP-1 (SP100030) at one-fourth to one-twentieth of their EC50 levels for 24 hours in BCWM.1 WM and TMD-8 ABC-DLBCL cells. (C) HCK mRNA levels were determined by TaqMan Gene Expression Assay following treatment by the combination of STAT3 (Galiellalactone), NF-kB (Triptolide [PG490]), and AP-1 (SR 11302) inhibitors at less than one-tenth to one-fortieth of their EC50 levels for 24 hours in BCWM.1 WM and TMD-8 ABC-DLBCL cells. Inhibitors used for each TF are labeled, and Comb indicates the combination of inhibitors to all 3 TFs. *P < .05; **P < .01; ***P < .001. DMSO, dimethyl sulfoxide.

Article Snippet: Native or HCK promoter-driven luciferase reporter transduced BCWM.1 or TMD8 cells were treated with inhibitors to transcription factors (TFs) STAT3 (STA-21; Selleck Chemicals, Houston, TX; Galiellalactone, Tocris Bioscience, Minneapolis, MN); AP1 (SP100030; SR 11302; Tocris Bioscience), and NF-κB (ACHP; Tocris Bioscience; QNZ; Triptolide [PG490]; Selleck Chemicals) for HCK transcription or promoter activity studies.

Techniques: Luciferase, Reporter Assay, Concentration Assay, Expressing, Labeling

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

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: Activin-A–induced STAT3 activation enhances CD73 expression in Th17 cells. (A) Representative FACS plots of Act-A–Th17 or Th17 cells showing pSTAT3 expression. Shaded histogram represents isotype control. Cumulative data are shown as mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (B) ChIP analyses demonstrating the binding of STAT3 on the Nt5e locus (site 2, +1,700 bp, Left) and on the Entpd1 promoter, at the SRE1 locus (−3,740 bp) (Right). Results are mean ± SEM; each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent experiments. (C) Act-A–Th17 cells or Th17 cells were cultured in the presence of STA-21. Gene expression was analyzed by qPCR and normalized to Gapdh and Polr2a. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of four independent experiments. (D) Cumulative data showing the percentages of CD39+, CD73+, and CD39+CD73+ among CD4+ T cells. Data are mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (E) IL-10 in culture supernatants. Each symbol represents the mean ± SEM of triplicate wells and corresponds to one of four independent experiments. Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: In some experiments, the AhR antagonist (CH-223191, 5 μM; Sigma-Aldrich), the CD73 antagonist (AMP-CP, 100 μM; Sigma-Aldrich), the Smad3 inhibitor (SIS3, 20 μm; Sigma-Aldrich), the neutralizing anti-ALK4 antibody (10 μg/mL; R&D), the STAT3 inhibitor (STA-21, 10 μM; Cayman Chemical) or vector (PBS) were administered in T cell cultures, as indicated.

Techniques: Activation Assay, Expressing, In Vitro, Binding Assay, Cell Culture

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

Article Title: Activin-A limits Th17 pathogenicity and autoimmune neuroinflammation via CD39 and CD73 ectonucleotidases and Hif1-α–dependent pathways

doi: 10.1073/pnas.1918196117

Figure Lengend Snippet: AhR drives activin-A–mediated up-regulation of CD73 and antiinflammatory genes in Th17 cells. (A) Representative immunoblots showing AhR and c-Maf protein levels in act-A–Th17 or Th17 cells. (B) Quantification of relative AhR and c-Maf expression is shown; TATA binding protein (TBP). Data are mean ± SEM; each symbol corresponds to one of four independent in vitro experiments. (C) ChIP analyses demonstrating the binding of AhR and c-Maf on the Nt5e locus. Data are mean ± SEM; each symbol represents one of four independent in vitro experiments. (D) ChIP analyses demonstrating the binding of AhR and c-Maf on the Entpd1 locus and (E) on the Il10 locus. Data are mean ± SEM; each symbol represents one of four independent in vitro experiments. (F) Sequential ChIP analysis demonstrating STAT3 and AhR cobinding on the Il10 conserved noncoding sequence-9 (−9.0 kb) locus. Data are mean ± SEM; each symbol corresponds to one of three independent in vitro experiments. (G) Act-A–Th17 or Th17 cells were differentiated, in the presence of the AhR antagonist, CH-223191 or control (DMSO). Gene expression was analyzed by qPCR and normalized to Gapdh and Polr2a. Each symbol represents the mean ± SEM of duplicate wells and corresponds to one of three independent in vitro experiments. Statistical analysis was performed by unpaired Student’s t test; *P < 0.05, **P < 0.01 and ***P < 0.001.

Article Snippet: In some experiments, the AhR antagonist (CH-223191, 5 μM; Sigma-Aldrich), the CD73 antagonist (AMP-CP, 100 μM; Sigma-Aldrich), the Smad3 inhibitor (SIS3, 20 μm; Sigma-Aldrich), the neutralizing anti-ALK4 antibody (10 μg/mL; R&D), the STAT3 inhibitor (STA-21, 10 μM; Cayman Chemical) or vector (PBS) were administered in T cell cultures, as indicated.

Techniques: Western Blot, Expressing, Binding Assay, In Vitro, Sequencing

Journal: Biomedicines

Article Title: IL-33 Enhances ACE2 Expression on Epidermal Keratinocytes in Atopic Dermatitis: A Plausible Issue for SARS-CoV-2 Transmission in Inflamed Atopic Skin

doi: 10.3390/biomedicines10051183

Figure Lengend Snippet: IL-33 enhanced ACE2 expression through ERK in keratinocytes. We performed immunofluorescent staining to measure the ACE2 expression in IL-33-treated keratinocytes. Small molecule inhibitors for PD98059 or STA21 (ERK and STAT3, respectively) were also pretreated. In the DMSO control, the fluorescence intensity of ACE2 (red color) was enhanced by IL-33 ( bottom left ). The fluorescence intensity decreased after PD98059 pretreatment ( bottom middle ). This phenomenon was not revealed after STA21 pretreatment ( bottom right ). Red: ACE2; blue: DAPI. The bar graph shows the quantitative data. In the DMSO control (white bar), the value of fluorescent area per cell increased under IL-33 stimulation. Moreover, under IL-33 stimulation (middle histogram), the value dramatically decreased after PD98059 pretreatment (gray bar), but not after STA21 pretreatment (black bar).

Article Snippet: The following cytokines and protein inhibitors were used: IL-33: PeproTech #200-17, 100 ng/mL; PD98059 (ERK 1/2 inhibitor): Sigma P215, 50 uM STA21 (STAT3 inhibitor): CAYMAN 14996, 250 nM.

Techniques: Expressing, Staining, Fluorescence

Journal: Biomedicines

Article Title: IL-33 Enhances ACE2 Expression on Epidermal Keratinocytes in Atopic Dermatitis: A Plausible Issue for SARS-CoV-2 Transmission in Inflamed Atopic Skin

doi: 10.3390/biomedicines10051183

Figure Lengend Snippet: IL-33 enhanced ACE2 expression through ERK in keratinocytes by flow cytometry. With a similar experimental design, we performed flow cytometry to measure ACE2 expression in IL-33-treated keratinocytes. Small molecule inhibitors for PD98059 or STA21 (ERK and STAT3, respectively) were pretreated in order to investigate the role of ERK or STAT3 in IL-33-induced ACE2 expression. The data showed that IL-33 induced a modest expression of ACE2, which was abrogated by both PD98059 and STA21. Interestingly, while IL-17 induced a minimally increased expression of ACE2, the pretreatment of STA21 potentiated the expression of ACE2 by IL-17.

Article Snippet: The following cytokines and protein inhibitors were used: IL-33: PeproTech #200-17, 100 ng/mL; PD98059 (ERK 1/2 inhibitor): Sigma P215, 50 uM STA21 (STAT3 inhibitor): CAYMAN 14996, 250 nM.

Techniques: Expressing, Flow Cytometry