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: 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: 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: 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

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Western blot analysis of MDA-MB-231 cell line stably transduced with a lentiviral vector encoding for control sh-RNA (CTR) or for sh-RNAs directed against human STAT3 (sh), serum starved and then stimulated for the indicated times with 5% wound fluids (WF). Vinculin expression was used as loading control. (B) qRT-PCR analysis of Bcl-2 and Survivin expression in MDA-MB-231 control cells (sh-no Target) or in STAT3 silenced clones (sh-STAT3). Cells were serum starved and then stimulated for the indicated times with wound fluids (WF). Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (C) Growth curve analysis of MDA-MB-231 control cells (sh-no Target) or STAT3 silenced clones (sh-STAT3). Cells (50×10 3 /well) have been seeded in complete medium (CM) on day 0, and then counted by Trypan Blue exclusion test, every day for 5 days. Two independent cell clones have been evaluated. Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (D) Growth curve analysis of MDA-MB-231 cell line in the presence of the indicated inhibitors. Cells (50×10 3 /well) have been seeded in complete medium (CM) on day 0, in the presence of S3I-201 (50 μM) or STA-21 (30 μM) or vehicle (CTR) and then counted by Trypan blue exclusion test, every day for 5 days. Fresh medium containing the inhibitor was replaced on day 3. Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (E) Same as in (C), but seeding cells in serum free medium supplemented with 3% wound fluids (WF). Three independent cell clones have been evaluated. Data represents the mean (± S.D.) of two independent experiments performed in triplicate. (F) Same as in (D), but seeding cells in serum free supplemented with 3% wound fluids (WF). Data represents the mean (± S.D.) of two independent experiments performed in triplicate.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Western Blot, Stable Transfection, Transduction, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Clone Assay

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Western blot analysis of MDA-MB-231 and MDA-MB-468 cell lines, serum starved and then stimulated for the indicated times with EGF (20 ng/mL) or 5% wound fluids (WF), as indicated. (B) Images show primary mammospheres formed by MDA-MB-231 cells. Control or STAT3-silenced (sh2 and sh3) cells were plated on poly-HEMA coated dishes in mammosphere growing medium supplemented with 5% wound fluids, in the presence of IL-6 blocking antibody (0.2μg/ml) or STAT3 inhibitors (S3I-201, 50 μM; Stattic, 10 μM; STA-21, 30 μM; and Galiellalactone, 12 μM), as indicated, and grown for ten days. (C) Same as in (B) but using MDA-MB-468 cells. STAT3 inhibitors were used as follows: S3I-201, 100 μM; Stattic, 10 μM; STA-21, 30 μM; Galiellalactone, 25 μM. (D) Graphs report the percent of mammosphere forming efficiency (MFE%) in MDA-MB-231 (left) and MDA-MB-468 (right) cells of the experiment described in (B) and (C). MFE was calculated as the ratio between the numbers of mammospheres counted/number of cells seeded, per well. (E) Graphs report the self-renewal in MDA-MB-231 (left) and MDA-MB-468 (right) cells treated with the inhibitors only during the second generation. Self-renewal was calculated as the ratio between number of secondary mammospheres/number of primary mammospheres. (F) Flow cytometry analysis of CD44 high CD24 low/neg stem cell-like subpopulation in MDA-MB-231 cells. Percent of CD44 high CD24 low/neg (Q4) and of CD44 high CD24 high (Q2) is reported in the plots.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Western Blot, Control, Blocking Assay, Flow Cytometry

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Graph reports the volume (mm 3 ) of primary tumors derived from injection of 2×10 6 MDA-MB-231 control (CTR) or STAT3 silenced (sh-STAT3) cells in thoracic mammary fat pads of nude mice (2 MFP/mouse) in 50 μl Matrigel/PBS (1:1). (B) Graph reports the time dependent appearance of primary tumors derived from injection of 2×10 6 MDA-MB-231 control (CTR) or STAT3 silenced (sh-STAT3) cells in the nude mouse thoracic mammary fat pads (2 MFP/mouse) in 50 μl Matrigel/PBS (1:1). (C) Graph reports the rate of tumor growth, independently from the time of appearance, in mice described in (A). Values are expressed as ratio of the tumor volume over the value of 20 mm 3 , considered as cut off. The red and the green lines represent the trend of growth of the MDA-MB-231 CTR and sh-STAT3, respectively. (D) Same as in (A), but injecting 2×10 5 MDA-MB-231 (CTR) or STAT3 silenced (sh-STAT3) cells, in place of 2×10 6 cells. (E) Same as in (B), but injecting 2×10 5 cells. (F) Same as in (C), but injecting 2×10 5 cells. In all graphs, statistical significance was calculated using the Student's t-test. One asterisk (*) indicates a p value ≤ 0.05, two asterisks (**) a p value ≤ 0.01 and three asterisk (***) a p value ≤ 0.005.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Derivative Assay, Injection, Control

Journal: Oncotarget

Article Title: Surgery-induced wound response promotes stem-like and tumor-initiating features of breast cancer cells, via STAT3 signaling

doi:

Figure Lengend Snippet: (A) Table reports the percentage of tumor take-rate, following injection of the indicated numbers of MDA-MB-231 CTR or sh-STAT3 clones, in the presence of Matrigel. The media between 2 to 3 different sh-STAT3 clones is reported. (B) Graph reports the disease free survival in the indicated cohorts of mice, after injection of decreasing number of MDA-MB-231 CTR or two different sh-STAT3 clones. Data are reported as percentage of mice that developed primary tumors during 12 weeks of follow-up. (C) The picture shows an example of primary masses excised from nude mice after surgery. It is evidenced the similar dimension of the tumors in the different groups. (D) Table reports the percentage of local recurrences formed by mice injected with MDA-MB-231 CTR cells or two different clones of sh-STAT3 after 8 weeks of follow up. (E) Graph reports the disease free survival in the indicated cohorts of mice, after removal of the primary tumor. Data are reported as percentage of mice that developed recurrent disease during 8 weeks of follow-up.

Article Snippet: In a subset of experiments, blocking antibody anti-IL6 (R&D Systems, 0.2μg/ml) or STAT3 inhibitors (S3I-201; STA-21; Stattic; Galiellalactone, purchased from Santa Cruz Biotechnology, Inc.) were added to the medium.

Techniques: Injection, Clone Assay