Journal: Cell Death & Disease

Article Title: CXCR4 uses STAT3-mediated slug expression to maintain radioresistance of non-small cell lung cancer cells: emerges as a potential prognostic biomarker for lung cancer

doi: 10.1038/s41419-020-03280-5

Figure Lengend Snippet: A Western blot analysis of phopho-STAT3 (Y705 and S727) in NSCLC cells (A549, H460, and A549/GR) transfected with the vectors described in the Figure. B Western blot (WB, upper) and RT-PCR (lower) analysis (left) and IR clonogenic survival assay (right) of A549/GR cells transfected with control siRNA (siCon) or siRNA against STAT3 (siSTAT3-1 and 3-2). C , G γ-H2AX foci assay of A549/GR (C) and A549/CXCR4 (upper) and H460/CXCR4 (lower) ( G ) cells. Cells were exposed to IR (6 Gy) and fixed at the indicated time points in the Figure. Representative ICC images of γ-H2AX (left) and quantification of the results (right). * P < 0.05, ** P < 0.01, *** P < 0.005. White bar: 20 μm. D , H Western blot analysis of A549/GR ( D ) and A549/CXCR4 (left) and H460/CXCR4 (right) ( H ). Cells exposed to IR (6 Gy) at the indicated time points transfected with control siRNA (siCon) or siRNA against Stat3 (siSTAT3-1). E , F Western bot analysis of p-STAT3 (S727 and Y705) (left) and IR clonogenic survival assay (right) in A549 ( E ) and H460 ( F ) cells transfected with control siRNA (siCon) or siRNA against STAT3 (siSTAT3-1 and siSTAT3-3).

Article Snippet: Antibody such as p-STAT3 (S727) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). p-STAT3 (Y705) and Slug were obtained from Cell Signaling Biotechnology (Denvers, MA).

Techniques: Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Clonogenic Cell Survival Assay, Control

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV results in activated phosphorylation of STAT3 on Tyr705 and diaphragm contractile dysfunction. Diaphragms from mechanically ventilated (MV) rats and unfed controls were analyzed. A ) Ex vivo force–frequency relationship from mechanically ventilated ( n =13) rats and unfed controls ( n =7). B ) Western blots for total STAT3 and phospho-STAT3 Y705 from a representative set of mechanically ventilated rats and unfed controls (1, 3, 6, and 9 h, n =5–6 rats/group; 18 h, n =6–8). C ) Messenger RNA levels of the STAT3 downstream target genes SOCS3 and Myf5 from mechanically ventilated ( n =9) rats and unfed controls ( n =10). Mechanical ventilation period of 18 h. Results are means ± sem . * P < 0.05; Student's t test.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Phospho-proteomics, Ex Vivo, Western Blot

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV increases Ser705 phosphorylation and mitochondrial accumulation of phospho-STAT3 (Ser727 and/or Tyr705/Ser727) within diaphragm muscle. A ) Diaphragms from unfed control rats ( n =8), MV-R548 rats ( n =9), or MV-Veh rats ( n =9) were analyzed by Western blot of total muscle lysate (representative animals depicted in blot) and quantitation. Correlation between increases in phospho-STAT3 S727 and phospho-STAT3 Y705 were determined. B ) Purified mitochondrial fractions (mito) and total lysates (total) were subjected to Western blot analysis for pSTAT3 S727 , pSTAT3 Y705 , total STAT3, GRIM-19, VDAC (mitochondrial marker), LDHA (cytoplasmic marker) and PCNA (nuclear marker). Unfed (U) controls, n = 10; MV-Veh (MV), n = 6; MV-R548, n = 7. Results are means ± sem . Mechanical ventilation period of 18 h. P values calculated by 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Phospho-proteomics, Control, Western Blot, Quantitation Assay, Purification, Marker

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: Proposed model for role of JAK signaling in VIDD. JAK signaling is activated by CMV and functions as a critical triggering mechanism upstream of STAT3 phosphorylation (Tyr705 and Ser705), mitochondrial dysfunction, ROS production, atrophy, and muscle weakness. Mechanical ventilation results in the mitochondrial accumulation of phospho-STAT3 (singly phosphorylated on Ser727 or doubly phosphorylated at both Ser727 and Tyr705). Import into mitochondria is facilitated through interaction of phospho-STAT3 with GRIM-19, a component of complex I of the ETC, and may directly affect mitochondrial function and ROS generation. Induction of various myogenic transcription factors and muscle-specific E3 ubiquitin ligases (MURF-1 and atrogin-1) and activation of calpain, caspase 9, and caspase 3 can contribute to muscle atrophy and proteolytic cleavage of myofilament proteins. Mitochondrial dysfunction may also lead to ROS-mediated modification of myofilament proteins in a manner that negatively affects contractile function.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Activation Assay, Modification

Journal: Cellular and Molecular Immunology

Article Title: A novel cytokine consisting of the p40 and EBI3 subunits suppresses experimental autoimmune arthritis via reciprocal regulation of Th17 and Treg cells

doi: 10.1038/s41423-021-00798-2

Figure Lengend Snippet: The p40-EBI3-Fc protein suppressed Th17 cells via regulation of IL-12Rb1 and gp130 receptor signaling. A Splenocytes from C57BL/6 mice were cultured with 10 ng/mL IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL). After 1 h, protein was isolated from the cultured cells. p -STAT3 Tyr705, STAT3, p -STAT5, STAT5, and GAPDH were analyzed by western blotting. Representative western blots from one of three independent experiments are shown (bottom panel); the results are depicted as the mean ± SD of three independent experiments per group (right). B Splenocytes isolated from C57BL/6 mice were stimulated with IL-6 in the presence or absence of the p40-EBI3-Fc protein (1 or 10 μg/mL) for 15 min. CD4 + T cells expressing pSTAT3 Tyr705, pSTAT3 Ser727, or pSTAT5 were then analyzed by flow cytometry. C A IL-12Rβ1 and gp130 overexpression vector was transfected into the Ba/F3 cell line using a P4 Primary cell 4D-nucleofector TM X kit. The proliferation of Ba/F3 cells was determined with a [ 3 H] thymidine incorporation assay under p40-EBI3-Fc (0.01–1 μg/mL) treatment. D IL-12Rβ1- and gp130-expressing Ba/F3 cells were stimulated with p40-EBI3-Fc for 1 h. p -STAT5, STAT5, and GAPDH in the cell lysates were analyzed using western blotting. Representative western blots from one of three independent experiments are shown; the results represent the mean ± SD of three independent experiments per group (right). E , F Alterations in IL-17 production in anti-CD3-stimulated murine splenic CD4 + T cells induced by p40-EBI3-Fc treatment and transfection with IL-12Rβ1-, gp130-, or WSX1-siRNA. Th17 cell expression was determined by FACS, and IL-17 expression was evaluated by ELISA. Bars show the mean ± SD of three independent experiments per group (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The permeabilized cells were stained with anti-p-STAT3 Y705-PE, anti-p-STAT3-S727-PE, or anti-p-STAT5-PE (BD Pharmingen) and subjected to flow cytometric analysis (CytoFLEX; Beckman Coulter, Fullerton, CA, USA).

Techniques: Cell Culture, Isolation, Western Blot, Expressing, Flow Cytometry, Over Expression, Plasmid Preparation, Transfection, Thymidine Incorporation Assay, Enzyme-linked Immunosorbent Assay

Journal: Clinical Cancer Research

Article Title: Reciprocal Regulation of c-Src and STAT3 in Non-Small Cell Lung Cancer

doi: 10.1158/1078-0432.ccr-09-0767

Figure Lengend Snippet: Fig. 4. Phosphorylated c-Src, STAT3, and downstream Src targets were measured in paired normal lung and NSCLC tumor tissues from patients who had previously undergone resection. A, tumors had higher mean c-Src activity, as indicated by decreased levels of inactive c-Src (pSrc Y527). Conversely, mean activated STAT3 (pSTAT3 Y705) was lower in tumor tissue than in normal lung. B, levels of inactive c-Src (pSrc Y527) correlated directly with activated STAT3 (pSTAT3 Y705) when analyzed as total levels of phosphorylated protein or ratio of phosphorylated protein to total protein. C, levels of phosphorylated FAK, p130Cas, and paxillin were inversely correlated with pSTAT3 Y705.

Article Snippet: Antibodies used in the Western blot analysis included c-Src and pSTAT3 S727 (Santa Cruz Biotechnology); pSrc Y419, pSTAT3 Y705, STAT3, pFAK Y861, Lyn, Yes, Bcl-XL, survivin, and STAT5 (Cell Signaling Technology); and β-actin (Sigma Chemical Company).

Techniques: Activity Assay

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: Aging (Albany NY)

Article Title: SLCO1B3 promotes colorectal cancer tumorigenesis and metastasis through STAT3

doi: 10.18632/aging.203502

Figure Lengend Snippet: Bioinformatics analysis of GSE163396. Microarray data analysis was performed to investigate the molecular mechanisms underlying the function of SLCO1B3 . ( A ) Heatmap of 286 differentially expressed genes from GSE163396 dataset (with 125 highly expressed genes and 161 lowly expressed genes). ( B – D ) GO functional enrichment and KEGG pathway analysis were performed based on DEGs from GSE163396 dataset. Partial results of the upregulated GO pathways were shown in panel B , the downregulated GO pathways were shown in panel C , and the KEGG pathway was illustrated in panel D . ( E ) Gene Set Enrichment Analysis (GSEA) revealed that most DEGs associated with the STAT3 signaling pathway were enriched in the SLCO1B3 gene. ( F ) The co-expression analysis revealed a positive association between the SLCO1B3 and STAT3 activation.

Article Snippet: The primary antibodies used in the experiments included an anti- OATP1B3 antibody from Proteintech Group Inc., (Wuhan, China; Catalog # 66381-1) and anti-STAT3 (Catalog # ab68153), anti-p-STAT3 (S727) (Catalog # ab32143), anti-MMP-2 (Catalog # ab92536), and anti-MMP9 (Catalog # ab58803) antibodies from Abcam (USA).

Techniques: Microarray, Functional Assay, Expressing, Activation Assay

Journal: Aging (Albany NY)

Article Title: SLCO1B3 promotes colorectal cancer tumorigenesis and metastasis through STAT3

doi: 10.18632/aging.203502

Figure Lengend Snippet: Ct-SLCO1B3 knockdown in HCT116 cells downregulates p-STAT3, MMP-2, and MMP-9. HCT116 cells were transiently transfected with si1-Ct-SLCO1B3 , si2-Ct-SLCO1B3 , or si-NC with or without IL-6 stimulation (50 ng/mL). The protein levels of Ct-OATP1B3 , p-STAT3, total STAT3, MMP-2, and MMP-9 were determined by western blot analysis. ( A ) Gel image. ( B ) Quantified protein levels without IL-6 stimulation. n=3, **P < 0.01 vs. si-NC. NC=si-NC, Si-1= si1-Ct-SLCO1B3 , Si-2= si2-Ct-SLCO1B3 .

Article Snippet: The primary antibodies used in the experiments included an anti- OATP1B3 antibody from Proteintech Group Inc., (Wuhan, China; Catalog # 66381-1) and anti-STAT3 (Catalog # ab68153), anti-p-STAT3 (S727) (Catalog # ab32143), anti-MMP-2 (Catalog # ab92536), and anti-MMP9 (Catalog # ab58803) antibodies from Abcam (USA).

Techniques: Transfection, Western Blot

Journal: Aging (Albany NY)

Article Title: SLCO1B3 promotes colorectal cancer tumorigenesis and metastasis through STAT3

doi: 10.18632/aging.203502

Figure Lengend Snippet: The effects of Ct-SLCO1B3 knockdown on CRC metastasis in vivo . ( A , B ) H&E staining of the liver and lung tissues showing metastatic nodules. ( C ) The expression of p-STAT3, STAT3, MMP-2, and MMP-9 in metastatic tumors by western blot analysis. **P < 0.01 vs. sh-Control.

Article Snippet: The primary antibodies used in the experiments included an anti- OATP1B3 antibody from Proteintech Group Inc., (Wuhan, China; Catalog # 66381-1) and anti-STAT3 (Catalog # ab68153), anti-p-STAT3 (S727) (Catalog # ab32143), anti-MMP-2 (Catalog # ab92536), and anti-MMP9 (Catalog # ab58803) antibodies from Abcam (USA).

Techniques: In Vivo, Staining, Expressing, Western Blot