Journal: International Journal of Molecular Medicine

Article Title: SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

doi: 10.3892/ijmm.2021.5037

Figure Lengend Snippet: SPHK1 binds to the novel protein NONO and thus affects STAT3 expression in bladder cancer cells. (A) Co-immunoprecipitation confirmed that SPHK1 bound to certain proteins in bladder cancer cells. (B) Mass spectrogram of the target protein NONO. (C) NONO and its downstream target STAT3 were validated via western blotting ( ** P<0.01 and *** P<0.001). SPHK1, sphingosine kinase 1; NONO, non-POU domain containing octamer binding; IP, immunoprecipitation; Vector, negative control; OE, overexpression.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: SPHK1 (cat. no. 10670-1-AP; ProteinTech Group, Inc.; 1:1,000), GAPDH (cat. no. AB0037; Shanghai Abways Biotechnology Co., Ltd.; 1:5,000;), NONO (cat. no. 385171; ZenBio, Inc.; 1:1,000), phosphorylated (p)-STAT3 (cat. no. 380906; ZenBio, Inc.; 1:1,000), Bax (cat. no. 50599-2-Ig; Cell Signaling Technology, Inc.; 1:1,000), Bcl2 (cat. no. 12789-1-AP; Cell Signaling Technology, Inc.; 1:1,000) and cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc.; 1:1,000).

Techniques: Expressing, Immunoprecipitation, Western Blot, Binding Assay, Plasmid Preparation, Negative Control, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

doi: 10.3892/ijmm.2021.5037

Figure Lengend Snippet: SPHK1 inhibition reverses the antiapoptotic effect in bladder cancer cells. (A) Inhibition efficiency of SPHK1 inhibitor FTY-720 as determined via western blotting ( *** P<0.001). (B) Inhibition of SPHK1 decreased the expression of NONO and p-STAT3 ( *** P<0.001). (C) Inhibition of SPHK1 decreased apoptosis in bladder cancer cells ( *** P<0.001). SPHK1, sphingosine kinase 1; NONO, non-POU domain containing octamer binding; p-, phosphorylated; OE, overexpression.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: SPHK1 (cat. no. 10670-1-AP; ProteinTech Group, Inc.; 1:1,000), GAPDH (cat. no. AB0037; Shanghai Abways Biotechnology Co., Ltd.; 1:5,000;), NONO (cat. no. 385171; ZenBio, Inc.; 1:1,000), phosphorylated (p)-STAT3 (cat. no. 380906; ZenBio, Inc.; 1:1,000), Bax (cat. no. 50599-2-Ig; Cell Signaling Technology, Inc.; 1:1,000), Bcl2 (cat. no. 12789-1-AP; Cell Signaling Technology, Inc.; 1:1,000) and cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc.; 1:1,000).

Techniques: Inhibition, Western Blot, Expressing, Binding Assay, Over Expression

Journal: International Journal of Molecular Medicine

Article Title: SPHK1 contributes to cisplatin resistance in bladder cancer cells via the NONO/STAT3 axis

doi: 10.3892/ijmm.2021.5037

Figure Lengend Snippet: Validation of SPHK1 and its corresponding proteins in the T24/DDP cell line. (A) DPP-resistant T24 bladder cancer cells were established and drug resistance was detected by performing the CCK-8 assay (1 µ g/ml: *** P<0.001 T24/DDP vs. T24; 2 µ g/ml: *** P<0.001 T24/DDP vs. T24). (B) Apoptosis was analyzed in T24 vs. T24/DDP (1 µ g/ml: *** P<0.001 T24/DDP vs. T24; 2 µ g/ml: *** P<0.001 T24/DDP vs. T24). (C and D) SPHK1, NONO and p-STAT3 were overexpressed in the T24/DDP cell line ( *** P<0.001). Following SPHK1 knockdown in the T24/DDP cell line, the results of (E and F) western blotting, (G) flow cytometry and (H) CCK-8 assay were similar to those reported in previous studies ( * P<0.05, ** P<0.01 and *** P<0.001). SPHK1, sphingosine kinase 1; DPP, cisplatin; CCK-8, Cell Counting Kit-8; NONO, non-POU domain containing octamer binding; p-, phosphorylated; OE, overexpression.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: SPHK1 (cat. no. 10670-1-AP; ProteinTech Group, Inc.; 1:1,000), GAPDH (cat. no. AB0037; Shanghai Abways Biotechnology Co., Ltd.; 1:5,000;), NONO (cat. no. 385171; ZenBio, Inc.; 1:1,000), phosphorylated (p)-STAT3 (cat. no. 380906; ZenBio, Inc.; 1:1,000), Bax (cat. no. 50599-2-Ig; Cell Signaling Technology, Inc.; 1:1,000), Bcl2 (cat. no. 12789-1-AP; Cell Signaling Technology, Inc.; 1:1,000) and cleaved caspase-3 (cat. no. 9661; Cell Signaling Technology, Inc.; 1:1,000).

Techniques: Biomarker Discovery, CCK-8 Assay, Knockdown, Western Blot, Flow Cytometry, Cell Counting, Binding Assay, Over Expression

Journal: Journal of Inflammation Research

Article Title: Anti-Inflammatory Regulatory Role of Signal Transducer and Activator of Transcription 3 Phosphorylation in Regulating Hypersensitivity Responses to Echinococcus granulosus Hydatid Cyst Fluid

doi: 10.2147/JIR.S509286

Figure Lengend Snippet: The inhibitory effect of different concentrations of inhibitors on STAT3 phosphorylation.

Article Snippet: The STAT3 inhibitors Stattic and JSI-124 were procured from Shanghai Haoyuan Chemexpress Co., Ltd. Antibodies for STAT3, phosphorylated p-STAT3 (S727), p-STAT3 (Y705), and ACTIN, as well as secondary antibodies, were purchased from Wuhan ABclonal Biotechnology Co., Ltd. High-resolution rapid electrophoresis liquid and ice-free rapid transfer liquid were obtained from Wuhan Servicebio Technology Co., Ltd. Immunoglobulin E (IgE) was purchased from Sigma-Aldrich, USA.

Techniques: Phospho-proteomics

Journal: International journal of oncology

Article Title: Salidroside inhibits migration, invasion and angiogenesis of MDA‑MB 231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2.

doi: 10.3892/ijo.2018.4430

Figure Lengend Snippet: Figure 2. SA inhibits angiogenesis in HUVECs. (A) In vitro angiogenesis assay revealed that SA inhibited angiogenesis in HUVEC cells (scale bars: 100 µm). (B) Graphical representation of the in vitro angiogenesis assay showing relative inhibition of tube formation. Statistical analyses were performed using Student's t-test. **P<0.01, ***P<0.001. (C) Western blot analysis revealed that VEGF and p‑STAT3 were inhibited in HUVECs treated with 40 µM SA for 24 h. (D) Relative protein expression levels of VEGF, p‑STAT3 and STAT3 were determined using densitometry; levels were normalized to β-actin. Data are representative of three independent experiments. Statistical analyses were performed using Student's t-test. ***P<0.001. HUVECs, human umbilical vein endothelial cells; p, phosphorylated; SA, salidroside; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor.

Article Snippet: Antibodies specific for phosphorylated (p)-STAT3 (Tyr705; sc-7993), STAT1 (sc-464), STAT3 (sc-8019), STAT5b (sc-1656), MMP3 (sc-6839), MMP9 (sc-21733), vascular endothelial growth factor (VEGF; sc-507), VEGF-R2 (sc-6251) and β-actin (sc-47778), together with secondary antibodies [goat antimouse (sc-2005), anti-rabbit (sc-2004) and donkey anti-goat (sc-2020) immunoglobulin G-horseradish peroxidase (HRP)], were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: In Vitro, Angiogenesis Assay, Inhibition, Western Blot, Expressing

Journal: International journal of oncology

Article Title: Salidroside inhibits migration, invasion and angiogenesis of MDA‑MB 231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2.

doi: 10.3892/ijo.2018.4430

Figure Lengend Snippet: Figure 3. SA inhibits EGFR/Jak2/STAT signaling activity and nuclear translocation, as well as the DNA-binding activity of STAT3. (A) Binding of SA (PubChem ID: 159278) to the ATP-binding domain of EGFR (Protein Data Bank ID: 2GS2), as determined by molecular docking using AutoDock Vina. (B) Western blot analysis revealed inhibition of EGFR activity and STAT3 signaling in MDA‑MB 231 cells following treatment with increasing concentrations of SA for 24 h. (C) MDA‑MB 231 cells were exposed to 10-40 µM salidroside for 24 h, and nuclear proteins were analyzed by western blotting, with TBP as a loading control. SA inhibited nuclear translocation of STAT3. (D) Gel shift analysis of nuclear extracts revealed that salidroside treatment inhibited the DNA-binding activity of STAT3 to the γ-interferon activation site element. EGFR, epidermal growth factor receptor; Jak2, Janus kinase 2; p, phosphorylated; SA, salidroside; STAT, signal transducer and activator of transcription; TBP, TATA-binding protein; VEGF, vascular endothelial growth factor; VEGF‑R2, VEGF receptor 2.

Article Snippet: Antibodies specific for phosphorylated (p)-STAT3 (Tyr705; sc-7993), STAT1 (sc-464), STAT3 (sc-8019), STAT5b (sc-1656), MMP3 (sc-6839), MMP9 (sc-21733), vascular endothelial growth factor (VEGF; sc-507), VEGF-R2 (sc-6251) and β-actin (sc-47778), together with secondary antibodies [goat antimouse (sc-2005), anti-rabbit (sc-2004) and donkey anti-goat (sc-2020) immunoglobulin G-horseradish peroxidase (HRP)], were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Activity Assay, Translocation Assay, Binding Assay, Western Blot, Inhibition, Control, Gel Shift, Activation Assay

Journal: International journal of oncology

Article Title: Salidroside inhibits migration, invasion and angiogenesis of MDA‑MB 231 TNBC cells by regulating EGFR/Jak2/STAT3 signaling via MMP2.

doi: 10.3892/ijo.2018.4430

Figure Lengend Snippet: Figure 4. STAT3 regulates MMP2 expression in MDA‑MB 231 cells upon SA treatment. (A) Targeted STAT3 silencing decreased MMP2 expression in MDA‑MB 231 cells. (B) Relative protein expression levels of p‑STAT3, STAT3 and MMP2 were determined using densitometry, and were normalized to β-actin. Data are representative of three independent experiments. Statistical analyses were performed using one-way analysis of variance. **P<0.01, ***P<0.001 compared with the control. (C) STAT3-overexpressing MDA‑MB 231 cells exhibited decreased p‑STAT3 and MMP2 protein expression following treatment with 40 µM SA. (D) Relative levels of p‑STAT3, STAT3, and MMP2 proteins were determined using densitometry, and normalized β-actin. Statistical analyses were performed using one-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001. Data are representative of three independent experiments. MMP2, matrix metalloproteinase 2; p, phosphorylated; SA, salidroside; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3.

Article Snippet: Antibodies specific for phosphorylated (p)-STAT3 (Tyr705; sc-7993), STAT1 (sc-464), STAT3 (sc-8019), STAT5b (sc-1656), MMP3 (sc-6839), MMP9 (sc-21733), vascular endothelial growth factor (VEGF; sc-507), VEGF-R2 (sc-6251) and β-actin (sc-47778), together with secondary antibodies [goat antimouse (sc-2005), anti-rabbit (sc-2004) and donkey anti-goat (sc-2020) immunoglobulin G-horseradish peroxidase (HRP)], were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Silencing of Annexin A1 suppressed the apoptosis and inflammatory response of preeclampsia rat trophoblasts

doi: 10.3892/ijmm.2018.3887

Figure Lengend Snippet: Knockdown of ANXA1 suppressed the JAK2/STAT3 pathway. The protein expression level of p-JAK2, JAK2, p-STAT3 and STAT3 were determined using western blot analysis. * P<0.05 and ** P<0.01, vs. empty vector group. ANXA1, Annexin A1; siRNA, small interfering RNA; JAK2, Janus kinase 2; STAT3, signal transducer and activator of transcription 3; p-, phosphorylated.

Article Snippet: Tris-buffered saline/Tween 20 buffer containing 5% skimmed milk was added to block the membranes at room temperature for 2 h. The corresponding primary antibodies were subsequently used to incubate the membranes overnight at 4°C, including anti-ANXA1 (cat. no. ab214486; 1:1,000; Abcam), anti-pro-caspase-3 (cat. no. ab32499; 1:1,000; Abcam), anti-cleaved-caspase-3 (cat. no. ab2302; 1:700; Abcam), anti-B-cell lymphoma-2 (Bcl-2; cat. no. ab692; 1:800; Abcam), anti-Bcl-2-associated X protein (Bax; cat. no. 2774; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-JAK2 (cat. no. ab108596; 1:600; Abcam), anti-phosphorylated (p)-JAK2 (cat. no. ab195055; 1:800; Abcam; anti-STAT3 (cat. no. ab119352; 1:1,000; Abcam), anti-p-STAT3 (cat. no. ab32143; 1:800; Abcam) and anti-GAPDH (cat. no. ab8245; 1:600; Abcam).

Techniques: Expressing, Western Blot, Plasmid Preparation, Small Interfering RNA

Journal: Frontiers in Aging Neuroscience

Article Title: Dl-3-n-Butylphthalide Promotes Remyelination and Suppresses Inflammation by Regulating AMPK/SIRT1 and STAT3/NF-κB Signaling in Chronic Cerebral Hypoperfusion

doi: 10.3389/fnagi.2020.00137

Figure Lengend Snippet: Dl-3-n-butylphthalide (NBP) suppresses inflammation induced after 2VO. (A) Western blot analysis of the expressions of TNF-a, p-STAT3 and NF-KB ( n = 6 in each group). (B) Quantitative analysis of protein levels of TNF-α. (C) Quantitative analysis of mRNA levels of TNF-α ( n = 6 in each group). (D) Quantitative analysis of protein levels of p-STAT3. (E) Quantitative analysis of protein levels of NF-KB. β-actin was used as an internal control. * p < 0.05, ** p < 0.01, *** p < 0.001, the model group vs. the sham group; # p < 0.05, ## p < 0.01, ### p < 0.001, the NBP60 group or NBP120 group vs. the model group; $ p < 0.05, $$ p < 0.01, the NBP60 group vs. NBP120 group; & p < 0.05, &&& p < 0.001, the model group sacrificed at 2 weeks vs. the model group sacrificed at 4 weeks. Values are expressed as mean ± SD. TNF-α, tumor necrosis factor-α. p-STAT3, phosphorylated signal transducers and activators of transcription 3. NF-κB, nuclear factor κB.

Article Snippet: Antibodies against the following proteins were used in this study: myelin basic protein (MBP; cat. no. ab40390), SIRT1 (cat. no. ab189494) and BrdU (cat. no. ab8152; all from Abcam, Cambridge, UK); glial fibrillary acidic protein (GFAP; cat. no. 16825-1-AP), oligodendrocyte transcription factor (Olig)2 (cat. no. 13999-1-AP), NF-κB (cat. no. 10745-1-AP), tumor necrosis factor (TNF)-α (cat. no. 17590-1-AP), and β-actin (cat. no. 20536-1-AP; all from Proteintech Biotechnology, Wuhan, China); phosphorylated (p-)STAT3 (cat. no. YP0250) and p-AMPKα1/2 (cat. no. YP0575; both from ImmunoWay, Plano, TX, USA).

Techniques: Western Blot, Control