Journal: PLoS ONE

Article Title: Deletion of the Innate Immune NLRP3 Receptor Abolishes Cardiac Ischemic Preconditioning and Is Associated with Decreased Il-6/STAT3 Signaling

doi: 10.1371/journal.pone.0040643

Figure Lengend Snippet: Effect of NLRP3 gene ablation on survival kinases and their phosphorylation status in preconditioned hearts analyzed at 5 min reperfusion following 35 min ischemia. PKC-ε (A), ERK (C), AMPK (E) and STAT3 (G), and their phosphorylation status (B, D, F, and H, respectively) are shown (n = 4 hearts per genotype). Mean ± SEM, *P<0.05 WT vs. NLRP3 −/− .

Article Snippet: To detect total proteins, the membranes were incubated at 4°C overnight with antibodies against STAT3 (1∶1000; Cell Signaling), AMPKα (1∶5000; Cell Signaling), p44/42 MAP kinase (1∶10000; Cell Signaling), against PKCε (1∶5000; Bio connect), and against α-tubulin (1∶20000; Sigma).

Techniques: Phospho-proteomics

Journal: BioMed Research International

Article Title: Leptin Modulates Norepinephrine-Mediated Melatonin Synthesis in Cultured Rat Pineal Gland

doi: 10.1155/2013/546516

Figure Lengend Snippet: Leptin signaling in cultured rat pineal gland. The glands were challenged with leptin (Lep 1 nM) alone and in association with norepinephrine (NE 1 μ M). Immunoblotting (IB) was performed against STAT3, a downstream protein of leptin signaling. α Tubulin is showed as internal control; values are expressed as arbitrary units. One-way ANOVA, followed by Bonferroni's post hoc test. * P < 0.05 versus control. n = 9 glands/group and each experiment was repeated 3 times.

Article Snippet: Antibodies against STAT3 and α Tubulin were purchased from Millipore (Billerica, MA, USA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), respectively.

Techniques: Cell Culture, Western Blot, Control

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Loss of Erbin induces STAT3 activation in cervical cancer cells. ( a , b ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% or 10% or no serum. The phosphorylations of FAK ( a ) and Src ( b ) were analyzed by western blot. ( c ) Parental HeLa (HeLa/WT), HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% or no serum. The phosphorylation of STAT3 was analyzed by western blot. The relative STAT3 phosphorylation levels were determined by densitometry and normalized with protein levels. ( d ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% serum in the plates coated with PolyHEMA. The phosphorylation of STAT3 was analyzed at the indicated time points. The relative STAT3 phosphorylation levels were determined by densitometry and normalized with protein levels. ( e ) HeLa/NC and HeLa/Erbin-sh cells were cultured in serum-free media. The phosphorylation of STAT3 was analyzed at the indicated time points. ( f ) HeLa/NC and HeLa/Erbin-sh cells were cultured in serum-free media overnight and then treated with 10 μℳ ISO. The phosphorylation of STAT3 was analyzed at the indicated time points. ** P <0.01.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Activation Assay, Cell Culture, Western Blot, Phospho-proteomics

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Loss of Erbin expression confers resistance of cervical cancer cells to anoikis in a STAT3-dependent manner. ( a , b ) HeLa cells were transfected with the plasmid expressing STAT3C (HeLa/STAT3C) or the empty vector (HeLa/Vector). The transfected cells were cultured in the medium containing 1% serum in the plates coated with PolyHEMA. After incubation for 48 and 72 h, the anoikis rates were determined by FACS. ( c , d ) HeLa/NC and HeLa/Erbin-sh cells cultured in suspension were treated with WP1066. The anoikis rates were determined by FACS. ( e , f ) HeLa/NC and HeLa/Erbin-sh cells under conventional culture were treated with WP1066. The apoptotic rates were determined by FACS. ( g , h ) HeLa/NC and HeLa/Erbin-sh cells were treated with WP1066, U0126 or PD98059. The anoikis rates were determined by FACS. ** P <0.01. DMSO, dimethyl sulfoxide; PI, propidium iodide.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Incubation, Suspension

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Erbin negatively regulates IL-6/STAT3 pathway. ( a ) HeLa/NC and HeLa/Erbin-sh cells under conventional culture were treated with 10 ng/ml IL-6. The activation of STAT3 was analyzed by western blot at the indicated time points. ( b ) HeLa/NC and HeLa/Erbin-sh cells were treated with 10 ng/ml IL-6 and then labeled with the anti-phoaphor-STAT3 antibody and Alexa fluor 549-labeled secondary antibody. Nuclei were stained with 1 μg/ml DAPI. Nuclear translocation of activated STAT3 was observed under a laser scanning confocal microscope. ( c ) HeLa/NC and HeLa/Erbin-sh cells grown in 24-well plates were transiently cotransfected by the STAT3 reporter and pRL-TK vectors. The transfected cells were treated with or without 10 ng/ml IL-6. The luciferase activities were measured using a dual luciferase assay kit. ( d , e ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% serum in the plates coated with PolyHEMA for 24 h and treated with or without 10 ng/ml IL-6. The phosphorylation of STAT3 was analyzed ( d ) and the luciferase activities were measured ( e ). ( f ) HeLa cells were treated with or without IL-6, and the expression of Erbin mRNA was analyzed by real-time reverse transcriptase–PCR (RT-PCR). ( g ) HeLa cells were transfected with pRc/CMV-Stat3C-Flag (HeLa/STAT3C) or the empty vector (HeLa/Vector). The expression of Erbin mRNA was examined by real-time RT-PCR. ( h ) The HeLa/STAT3C and HeLa/Vector cells were transfected with Erbin siRNA or control siRNA. The expression of Erbin protein was examined by western blot. ( i ) HeLa cells were starved overnight and then treated with AG490. IL-6 was added into the cell culture after 1 h of the treatment. The phosphorylation of STAT3 was analyzed by western blot at the indicated time points. ** P <0.01.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Activation Assay, Western Blot, Labeling, Staining, Translocation Assay, Microscopy, Transfection, Luciferase, Cell Culture, Phospho-proteomics, Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Quantitative RT-PCR, Control

Journal: Oral oncology

Article Title: Co-targeting ALK and EGFR parallel signaling in oral squamous cell carcinoma.

doi: 10.1016/j.oraloncology.2016.05.007

Figure Lengend Snippet: Fig. 4. Western blot analysis of EGFR signaling components in OSCC cell lines treated for six hours with Gefitinib (500 nM), TAE684(500 nM), or both. Panel A: HSC3 cells treated with Gefitinib demonstrate a reduced ratio of phosphorylated EGFR (p-EGFR) to total EGFR. An additive reduction of p-EGFR/EGFR is seen with a combination treatment (Combo) of TAE684 and Gefitinib. The ratio of phosphorylated AKT (p-AKT) to total AKT is significantly reduces with individual treatments and abolished with combination treatments. No change in the ratio of phosphorylated ERK1/2 (p-ERK1/2) to total ERK1/2 is seen. Lone treatment with the ALK inhibitor, TAE684, significantly induces activation of STAT3 as reflected in the ratio of phosphorylated STAT3 (p-STAT3) to total STAT3. This induction is reversed with combination treatments of TAE684 and Gefitinib. Panel B: Cal27 cells treated with Gefitinib demonstrate no significant changes in the ratio of p-EGFR to total EGFR. Gefitinib alone abolished p-AKT and TAE684 alone significantly reduced the ratio of p-AKT to total AKT. Similarly, combination treatment of Gefitinib and TAE684 (Combo) abolished p-AKT and no change in the ratio of p- ERK1/2 to total ERK1/2 is seen in Cal27 cells. Although a trend is seen with an increase in the p-STAT3 to total STAT3 ratio is seen with all treatments, no significant changes were detected. *p < 0.05, **p < 0.01, ***p < 0.001 denote significance between treatments.

Article Snippet: Rabbit monoclonal antibodies against STAT3 (1:2000; #12640), p-STAT3(Tyr705) (1:4000; #9145) and ERK1/2 (1:2000; #4695) were used (Cell Signaling; Danvers, MA).

Techniques: Western Blot, Activation Assay

Journal: Frontiers in Oncology

Article Title: 5-Methoxytryptophan Sensitizing Head and Neck Squamous Carcinoma Cell to Cisplatitn Through Inhibiting Signal Transducer and Activator of Transcription 3 (STAT3)

doi: 10.3389/fonc.2022.834941

Figure Lengend Snippet: Inhibitory effect of the combination treatment of Cisplatin (CDDP) and 5-methoxytryptophan (5-MTP) on proliferation involved in the STAT3 signaling pathway. SCC4 and SCC25 cells were treated with CDDP, 5-MTP, or both at indicated concentrations for 6 h (A, B) or 24 h (C, D) . Total cell lysates were extracted at the indicated time points and subjected to western blot with relevant antibodies. GAPDH served as an equal loading control in western blot. Asterisks indicate significant difference in drug-treated cells at indicated concentrations singly in comparison with DMSO-treated cells. *P < 0.05.

Article Snippet: The target proteins were probed with specific antibodies against p-STAT3 (ABclonal; 1:1000, AP0705), p-Akt (ABclonal; 1:1000, AP0637), p-p65 (Cell Signaling; 1:1000, 3033T), p-p38 (ABclonal; 1:1000, AP0526), PCNA (GeneTex; 1:3000, GTX100539), GAPDH (GeneTex; 1:10000, GTX100118).

Techniques: Western Blot, Control, Comparison

Journal: Frontiers in Oncology

Article Title: 5-Methoxytryptophan Sensitizing Head and Neck Squamous Carcinoma Cell to Cisplatitn Through Inhibiting Signal Transducer and Activator of Transcription 3 (STAT3)

doi: 10.3389/fonc.2022.834941

Figure Lengend Snippet: Additional inhibitory effect of the combination treatment of Cisplatin (CDDP) and 5-methoxytryptophan (5-MTP) in 4NQO-induced tongue oral squamous cell carcinoma (OSCC) mouse model. (A) Morphological observation of the DMSO- or drug-treated group in 4NQO-induced cancer mouse model. (B) Digital outlines of representative tongues of DMSO- or drug-treated mice. Asterisks indicate significant difference in drug-treated group singly in comparison with 4NQO-treated group. *P < 0.05 (C) hematoxylin–eosin or immunohistochemical staining with PCNA and p-STAT3 antibodies of tongue cancer with DMSO or drug treatment.

Article Snippet: The target proteins were probed with specific antibodies against p-STAT3 (ABclonal; 1:1000, AP0705), p-Akt (ABclonal; 1:1000, AP0637), p-p65 (Cell Signaling; 1:1000, 3033T), p-p38 (ABclonal; 1:1000, AP0526), PCNA (GeneTex; 1:3000, GTX100539), GAPDH (GeneTex; 1:10000, GTX100118).

Techniques: Comparison, Immunohistochemical staining, Staining

Journal: Nutrients

Article Title: Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway

doi: 10.3390/nu14132669

Figure Lengend Snippet: Hydroxycitric acid promotes AMPK phosphorylation in CML cells. CML cell lines were treated for 24 h with different concentrations of HCA. An equal amount of protein from each condition was separated on SDS PAGE (12% gel) and an immunoblot was performed using specific antibodies against pT172 AMPK and total AMPK. Vinculin was used as an internal control. ( A ) K562 ( B ) MEG-01 ( C ) KYO-1 ( D ) SHK-1. For the K562, MEG-01, KYO-1, and SHK-1, samples were loaded in duplicate. One was probed with total AMPK and the another one was probed with pT172 AMPK. Upper vinculin is referred to as pAMPK, and lower vinculin is referred to as the total AMPK. The bar graph beside each figure panel reflects the band intensity evaluated as optical density and represented as fold change for treated vs. untreated cells normalized for vinculin. ** p < 0.01, * p < 0.05 treated vs. untreated cells.

Article Snippet: After washing them thoroughly, immune complexes were eluted from the beads by boiling them in Laemmili buffer and were separated on SDS-PAGE (12% gels) and analyzed by Western blot analysis with antibody against AMPK (Cell-Signaling, Danvers, MA, USA; #2523S; 1:1000) and ACLY (Cell-Signaling, Danvers, MA, USA, #4332S; 1:1000).

Techniques: Phospho-proteomics, SDS Page, Western Blot, Control

Journal: Nutrients

Article Title: Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway

doi: 10.3390/nu14132669

Figure Lengend Snippet: AMPK interacts with ACLY. ( A ) Co-immunoprecipitation of AMPK and ACLY in K562 cells. Endogenous AMPK was immunoprecipitated with antibody against total AMPK followed by Western blotting with anti-AMPK and anti-ACLY (upper panel). Bar graph showing the ratio of optical density of immunoprecipitated (IP) band of ACLY and AMPK after normalization with the respective input (lower panel). ( B ) Co-immunoprecipitation of AMPK and ACLY in K562 cells upon treatment with HCA (upper panel). Bar graph of optical density of immunoprecipitated ACLY and AMPK in each condition normalized to respective input band (left lower panel). Ratio of optical density of immunoprecipitated (IP) band of ACLY and AMPK after normalization with the control (right lower panel).

Article Snippet: After washing them thoroughly, immune complexes were eluted from the beads by boiling them in Laemmili buffer and were separated on SDS-PAGE (12% gels) and analyzed by Western blot analysis with antibody against AMPK (Cell-Signaling, Danvers, MA, USA; #2523S; 1:1000) and ACLY (Cell-Signaling, Danvers, MA, USA, #4332S; 1:1000).

Techniques: Immunoprecipitation, Western Blot, Control