Journal: The Journal of Clinical Investigation

Article Title: STING agonist 8803 reprograms the immune microenvironment and increases survival in preclinical models of glioblastoma

doi: 10.1172/JCI175033

Figure Lengend Snippet: ( A ) Schema of the treatment of immunocompetent mice that underwent i.c. implantation of GL261 glioma cells. Seven days after GL261 implantation, mice were treated with WP1066 (60 mg/kg) by oral gavage (o.g.) 3 times per week (M/W/F) for 3 weeks. On day 7, mice were treated intratumorally with the STING agonist 8803 (5 μg). The survival rate of C57BL/6J mice was estimated by the Kaplan-Meier method. Control: 11 mice (median survival [MS]: 18 days), WP1066: 8 mice (MS: 21 days), 8803: 8 mice (MS: 143.5 days); 4 long-term survivor), WP1066 + 8803 agonist: 7 mice (MS: 25 days). Statistics (log-rank test): control versus WP1066 P = 0.0047; control versus 8803 P < 0.0001; control versus WP1066 + 8803 P = 0.0002; WP1066 versus WP1066 + 8803 P = 0.02; 8803 versus WP1066 + 8803 P = 0.0005; 8803 versus WP1066 P < 0.0001. ( B ) In vitro luciferase expression assay for the induction of IFN responses. Various concentrations of the STING agonist 8803 were used to induce the luciferase expression in the top panels. A physiological (2 μM) and a high dose (5 μM) of WP1066 was used in combination. Direct cellular cytotoxicity was measured during the above experimental conditions (lower panels). At a WP1066 concentration of 5 μM, there was a decrease in IFN activity, which was not attributed to cell viability. ( C ) WP1066 decreases STING protein expression in THP-1 cells in a dose-dependent manner. THP-1 cells were treated with the indicated concentrations of WP1066. Cells were collected and analyzed by Western blotting. ( D ) Ubiquitination assay of STING in THP-1 cells indicates that WP1066 induces STING ubiquitination in a dose-dependent manner. MG132, proteasome inhibitor used in all conditionsl; IB, immunoblotting; UB, ubiquitination blotting; IP, immunoprecipitation.

Article Snippet: WP1066 , which blocks p-STAT3 , was supplied in-house and from Medchem Express (HY-15312).

Techniques: Control, In Vitro, Luciferase, Expressing, Concentration Assay, Activity Assay, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: STING agonist 8803 reprograms the immune microenvironment and increases survival in preclinical models of glioblastoma

doi: 10.1172/JCI175033

Figure Lengend Snippet: ( A ) Schema of the treatment of immunocompetent mice that underwent i.c. implantation of either GL261 or CT-2A glioma cells. ( B ) The survival rate estimated by the Kaplan-Meier method of C57BL/6J mice implanted with GL261 and CT-2A. For the GL261 survival: control: 10 mice (median survival [MS]: 25 days), WP1066: 10 mice (MS: 30 days), 8803: 14 mice (MS: 30 days; 5 long-term survivors), WP1066 + 8803: 16 mice (MS: 71 days; 8 long-term survivors). Statistics (log-rank test): control versus WP1066 P = 0.04; control versus 8803 P = 0.01; control versus WP1066 + 8803 P < 0.0001; WP1066 versus WP1066 + 8803 P = 0.006; 8803 versus WP1066 + 8803 P = 0.32. For the CT-2A survival: control: 10 mice (MS: 28 days), WP1066: 9 mice (MS: 28 days), 8803: 14 mice (MS: 50 days; 7 long-term survivors), WP1066 + 8803: 16 mice (MS: 48 days; 8 long-term survivors). Statistics (log-rank test): control versus WP1066 P = 0.57; control versus 8803 P < 0.0001; control versus WP1066 + 8803 P < 0.0001; WP1066 versus WP1066 + 8803 P < 0.001; 8803 versus WP1066 + 8803 P = 0.95. ( C ) Schema of the treatment of immunocompetent mice that underwent i.c. implantation of either CT-2A or QPP8v (subclone) glioma cells. QPP8v tumors received 8803 (5 μg/mouse i.c. on days 7 and 17), anti–PD-1 (25 μg i.c. on days 7 and 17), and vehicle control (PBS) or anti–PD-1 (250 μg i.p. on days 7, 10, and 13) in single and combination therapies. ( D ) The survival rate estimated by the Kaplan-Meier method of C57BL/6J mice implanted with CT-2A and QPP8. For CT-2A: IgG: 10 mice (MS: 30 days); anti–PD-1: 10 mice (MS: 35 days), 8803: 10 mice (MS: 56 days, 4 long-term survivors); 8803 + anti–PD-1: 10 mice (MS: undefined, 8 long-term survivors); 8803 + IgG: 10 mice (MS: 65.5 days, 5 long-term survivors). Statistics (log-rank test): IgG versus anti–PD-1 P = 0.04; IgG versus 8803 P < 0.01; IgG versus 8803 + anti–PD-1 P < 0.0001; anti–PD-1 versus 8803 + anti–PD-1 P < 0.001; 8803 versus 8803 + anti–PD-1 P = 0.04; 8803 + IgG versus 8803 + anti–PD-1 P = 0.12. For QPP8: PBS: 10 mice (MS: 47 days); anti–PD-1: 10 mice (MS: 55 days); 8803: 10 mice (MS: 76 days, 4 long-term survivors); 8803 + anti–PD-1 (i.p.): 10 mice (MS: 95.5 days, 3 long-term survivors); 8803 + anti–PD-1 (i.c.): 10 mice (MS: 111 days, 5 long-term survivors). Statistics (log-rank test): PBS versus anti–PD-1 P = 0.164; PBS versus 8803 P < 0.0001; PBS versus 8803 + anti–PD-1 (i.p.) P = 0.0006; PBS versus 8803 + anti–PD-1 (i.c.) P < 0.0001; anti–PD-1 versus 8803 P < 0.0001; anti–PD-1 versus 8803 + anti–PD-1 (i.p.) P = 0.002; anti–PD-1 versus 8803 + anti–PD-1 (i.c.) P < 0.0001; 8803 versus 8803 + anti–PD-1 (i.c.) P = 0.57; 8803 versus 8803 + anti–PD-1 (i.p.) P = 0.85. ( E ) Multiplexed sequential immunofluorescence images of untreated CT-2A gliomas ( n = 3). Forty-eight hours after either the first (day 9) or second dose (day 16) of 8803, animals were euthanized and the brains were imaged for the following ( n = 3/group): DAPI (dark blue), GFAP (light blue), CD31 (cyan blue), STING (red), p-IRF3 (pink), CD4 + (yellow), and CD8 + (white) T cells. White boxes outline the portion shown at higher magnification in the right column of images. Scale bars: 500 μm (left 3 columns) and 50 μm (right column).

Article Snippet: WP1066 , which blocks p-STAT3 , was supplied in-house and from Medchem Express (HY-15312).

Techniques: Control, Immunofluorescence

Journal: Oncology reports

Article Title: NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells.

doi: 10.3892/or.2016.4761

Figure Lengend Snippet: Figure 3. NF-YA increases the expression of VEGF and activity of STAT3. (A) NF-YA, VEGF and β-actin protein levels were detected by western blot analysis in vector control or NF-YA stably overexpressed A375P/Malme-3M cells (left panel); relative mRNA expression of VEGF in vector control or NF-YA stably overexpressed A375P/Malme-3M cells by real-time quantitative PCR (left panel), *P<0.003 compared with the control group. (B) STAT3, pY-STAT3 (tyrosine phosphorylated STAT3 at the 705 residue) and β-actin protein levels were detected by western blot analysis in vector control or NF-YA stably overexpressed A375P/Malme-3M cells. (C) Immunofluorescence staining showing expression of pY-STAT3 (red) in vector control or NF-YA stably overexpressed A375P cells. Nuclei were stained with DAPI (blue).

Article Snippet: STAT3 inhibitor WP1066 (working concentration: 5 μM) was purchased from Selleck Chemicals (cat no. S2796; Houston, TX, uSA).

Techniques: Expressing, Activity Assay, Western Blot, Plasmid Preparation, Control, Stable Transfection, Real-time Polymerase Chain Reaction, Residue, Immunofluorescence, Staining

Journal: Oncology reports

Article Title: NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells.

doi: 10.3892/or.2016.4761

Figure Lengend Snippet: Figure 4. NF-YA upregulated VEGF in a STAT3-dependent manner. (A) NF-YA, STAT3, pY-STAT3, VEGF and β-actin protein levels were detected by western blot analysis in vector control or NF-YA stably overexpressed (or NF-YA stably overexpressed plus STAT3 inhibitor WP1066 treatment) A375P/Malme-3M cells. (B) EZH2 and β-actin protein levels were detected by western blot analysis in vector control or NF-YA stably overexpressed A375P/Malme-3M cells (upper panel). Relative mRNA expression of VEGF in vector control or NF-YA stably overexpressed A375P/Malme-3M cells by real-time quantitative PCR (low panel), *P<0.004 compared with the control group. (C) NF-YA, EZH2, STAT3, pY-STAT3, VEGF and β-actin protein levels were detected by western blot analysis in scramble control or NF-YA stably knockdown (sh-1-N, sh-2-N) A375-M2 cells.

Article Snippet: STAT3 inhibitor WP1066 (working concentration: 5 μM) was purchased from Selleck Chemicals (cat no. S2796; Houston, TX, uSA).

Techniques: Western Blot, Plasmid Preparation, Control, Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Knockdown

Journal: Oncology reports

Article Title: NF-YA promotes invasion and angiogenesis by upregulating EZH2-STAT3 signaling in human melanoma cells.

doi: 10.3892/or.2016.4761

Figure Lengend Snippet: Figure 5. The role of EZH2 in NF-YA induced VEGF expression and STAT3 activation. (A) Immunoprecipitation of STAT3 protein using antibodies for STAT3 (IP), or a non-specific immunoglobulin G (IgG) from scramble control or NF-YA stably knockdown (sh-1-N) A375-M2 cells, followed by immunoblots with the indicated antibodies. Methy-K, means rabbit anti-methylated lysine antibody; input indicate 5% of pre-immunoprecipitated samples. (B) NF-YA, EZH2, STAT3, pY-STAT3, VEGF and β-actin protein levels were detected by western blot analysis in scramble control or EZH2 stably knockdown (sh-1-E, sh-2-E) A375P cells (left panel); immunoprecipitation of STAT3 protein using antibodies for STAT3 (IP), or a non-specific immunoglobulin G (IgG) from scramble control or EZH2 stably knockdown (sh-2-E) A375P cells, followed by immunoblots with the indicated antibodies. Methy-K, means rabbit anti- methylated lysine antibody; input indicate 5% of pre-immunoprecipitated samples (middle panel); NF-YA, STAT3, pY-STAT3, VEGF and β-actin protein levels were detected by western blot analysis in vector control and NF-YA stably overexpressed A375P cells with EZH2 stably knockdown (sh-2-E) (right panel). (C) The vector control or NF-YA stably overexpressed A375P-sh-2-E cells were seeded in a Matrigel‑coated upper chamber. Cells invaded through the Matrigel after incubation for 24 h was detected by H&E staining and quantified (lower panel). Data expression was analyzed by quantification of six randomly selected fields. A375P-sh-2-E, means A375P cells with EZH2 stably knockdown (sh-2-E). (D) HUVECs cells were plated in the upper chamber coated with Matrigel in Transwell filters (0.4 µm). The vector control or NF-YA stably overexpressed A375P-sh-2-E cells were grown in the lower chamber. Cultured for 3 days, the tube-like structures that formed in the gel were photographed (magnification, x100).

Article Snippet: STAT3 inhibitor WP1066 (working concentration: 5 μM) was purchased from Selleck Chemicals (cat no. S2796; Houston, TX, uSA).

Techniques: Expressing, Activation Assay, Immunoprecipitation, Control, Stable Transfection, Knockdown, Western Blot, Methylation, Plasmid Preparation, Incubation, Staining, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: High temperature suppressed SSC self-renewal through S phase cell cycle arrest but not apoptosis

doi: 10.1186/s13287-019-1335-5

Figure Lengend Snippet: The JAK-STAT signalling pathway is involved in the inhibition of spermatogonial stem cell regeneration induced by high temperature. a The expression of the negative feedback inhibitor Socs3 in the JAK-STAT signalling pathway was upregulated 2 h after heat shock treatment, while the phosphorylation of STAT3 was reduced. The phosphorylation of Akt in the PI3K-Akt signalling pathway, which was regulated by the JAK-STAT signalling pathway, was found to be reduced. b Inhibition of the JAK/STAT signalling pathway by WP1066 in non-treated SSCs induced a decrease in proliferation. The asterisk indicates a significant difference from the control group

Article Snippet: SSCs were plated at a density of 2 × 10 3 cells/well in 96-well plates, cultured for 2 h, washed with medium and then cultured for an additional 20 h in SSC medium in the presence or absence of 1.25 μM, 2.5 μM or 5 μM WP1066 (A JAK-STAT signalling pathway inhibitor; A4140, APExBIO).

Techniques: Inhibition, Expressing, Phospho-proteomics, Control

Journal: Cell Communication and Signaling : CCS

Article Title: PI3K-AKT, JAK2-STAT3 pathways and cell–cell contact regulate maspin subcellular localization

doi: 10.1186/s12964-021-00758-3

Figure Lengend Snippet: Antibodies and inhibitors employed in this study

Article Snippet: AdooQ Biosciences (#A11795) , WP1066 , JAK2/STAT3 , 10 μM.

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: PI3K-AKT, JAK2-STAT3 pathways and cell–cell contact regulate maspin subcellular localization

doi: 10.1186/s12964-021-00758-3

Figure Lengend Snippet: EGF-induced maspin nuclear accumulation depends on the PI3K-Akt and JAK2-STAT3 pathways A Starved MCF-10A cells were pretreated with inhibitors of EGFR (Gefitinib), PI3K (Wortmannin), pan-PKC (Go6983), JAK2/STAT3 (WP1066), H/K-Ras (FTI-277) and Src family kinases (PP2) (except for the upper row), for 30 min followed by 20 ng/ml EGF treatment for additional 1 h. Maspin localization was analyzed by immunofluorescence with anti-maspin antibody. Nuclei were stained with DAPI. B Cells were quantified based on the criteria shown next to the graph * p < 0.05 (Chi-square test). C The experiment described in A was repeated and images were acquired in a higher resolution. JAK2/STAT3 inhibition by WP1066 leads to a junctional discontinuity pattern, whereas PI3K inhibition by Wortmannin results in continuous maspin distribution across cell–cell junctions (arrows). Images are representative of at least three independent assays. Scale bar: 20 µm

Article Snippet: AdooQ Biosciences (#A11795) , WP1066 , JAK2/STAT3 , 10 μM.

Techniques: Immunofluorescence, Staining, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

doi: 10.3390/ijms22010460

Figure Lengend Snippet: Development rates of mouse embryos at E3.5 and transcription activities of the Gzmg promoter by culturing with STAT3 inhibitors.

Article Snippet: Inc., Santa Cruz, CA, USA) [ ] or 5-μM WP1066 (cat. no. ALX-270-483; Enzo Life Science Inc., New York, NY, USA) [ ], and KSOM-AA medium (cat. no. MR-106; Sigma-Aldrich, St. Louis, MO, USA) were changed every 24 h. Embryos were observed by light microscopy every day to determine the developmental stages of the preimplantation embryos.

Techniques: Activity Assay, Fluorescence