Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Identification of osthole binding proteins. a Chemical structure of osthole intermediate and Biotin-labeled osthole. b Viability of MDA-MB-231 cells exposed to biotin-labeled osthole as determined by MTT assay. c A schematic illustrating the steps for identifying Bio-osthole binding to proteins fabricated on a microarray. d Representative image of an experimental microarray [Blue = negative control, red = positive control, yellow = positive spot]. e Magnified image of Bio-osthole binding to recombinant STAT3 protein spot on the microarray Signal to noise ratio (SNR) value is shown

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Binding Assay, Labeling, MTT Assay, Microarray, Negative Control, Positive Control, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Osthole inhibits STAT3 phosphorylation in TNBC cells. a MDA-MB-231 and BT-549 cells were treated with 200 μM osthole for the indicated times, and levels of P-STAT3 were determined by Western blot analysis. GAPDH and STAT3 were used as internal control. b MDA-MB-231 and BT-549 cells were exposed to osthole at indicated concentrations for 24 h or 12 h, respectively. P-STAT3 levels were determined by immunoblotting. GAPDH and STAT3 were used as internal control. c Cells were pretreated with 200 μM osthole for 24 h (MDA-MB-231) or 12 h (BT-549) and then stimulated with IL-6 (50 ng/mL) for 30 mins. STAT3 phosphorylation was determined by western blot. d Immunofluorescence staining of cells showing distribution of P-STAT3 (red) in MDA-MB-231 cells. DAPI was used as counter stain. e MDA-MB-231 cells were pretreated with osthole for 24 h before exposure to IL-6 (50 ng/mL) for 30 min. Nuclear extracts were subjected to P-STAT3 and STAT3 immunoblotting. Lamin B was used as loading control

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: STAT3 overexpression rescued osthole-mediated cytotoxic effects in MDA-MB-231 cells. a Representative western blot showing levels of STAT3 and P-STAT3 in MDA-MB-231 cells following transfection with STAT3 plasmid [Control plasmid = control vehicle vector, STAT3 = STAT3 plasmid]. b Quantification of STAT3 protein levels from panel A. [* P < 0.05 compared to control plasmid (V)]. c STAT3 overexpressing cells and vector control transfected cells were exposed to 200 μM osthole for 48 h, and apoptotic cells were determined by Annexin V/PI staining. d Quantification of annexin V/PI staining showing the percentage of apoptotic cells from panel C [*** P < 0.001]. (E) STAT3 overexpressing cells and vector control transfected cells were exposed to 200 μM osthole for 36 h, and representative histograms about G2/M cell cycle phase in cells were determined by flow cytometric analysis [* P < 0.05]

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Osthole inhibits MDA-MB-231 xenograft growth in vivo. a Tumor volume in vehicle- and osthole-treated mice. MDA-MB-231 cells were injected in the flanks of mice and tumors were allowed to develop for approximately 8 d (50–150 mm 3 ). Mice bearing MDA-MB-231 xenografts received osthole at 100 or 200 mg/kg interperitoneally [* P < 0.05]. b Images of resected tumor tissues at day 48. Two mice (one in 100 mg/kg osthole group and the other in 200 mg/kg osthole group did not have a visible tumor after 48 d treatment. c Tumor weights determined on day 48 [* P < 0.05 compared to vehicle control]. d Western blot analysis of P-STAT3 levels in resected tumor specimens. GAPDH was used as loading control. e Immunohistochemical staining of tumor sections for cell proliferation marker Ki-67, apoptosis markers Bcl-2 and cleaved caspase-3, and cell cycle markers MDM2, and CDC2. Representative images are shown

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: In Vivo, Injection, Western Blot, Immunohistochemical staining, Staining, Marker

Journal: Veterinary Research

Article Title: Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses

doi: 10.1186/s13567-014-0118-3

Figure Lengend Snippet: Primer and probe sequences for quantitative reverse transcription PCR assays

Article Snippet: Primary chicken embryo cells in 6-well culture plates (Costar) were transiently transfected with constitutively active mouse STAT-3 expression plasmid [Stat3-C Flag pRc/CM V, plasmid 8722, Addgene, USA] or empty pRc/CMV vector (Invitrogen) using TransIT-LT1 reagent (Mirus Bio, Cambridge, UK).

Techniques: Reverse Transcription, Sequencing

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: Reversible reduction in IL-6-induced STAT3 signaling in response to mild oxidative stress. A, HepG2 cells were serum starved for 3–4 h and then left untreated or treated with PDTC (100 μm, 2 h) or diamide (500 μm, 30 min), followed by stimulation with IL-6 (20 ng/ml) for 20 min at 37 C. B, Cells were pretreated with diamide (500 μm) for 30 min, washed, and incubated with fresh medium for 0, 5, 15, and 45 min before the addition of IL-6 for 15 min. A and B, Cells were lysed and analyzed by Western blot with an antibody raised against pY-STAT3 (top panels). ERK1/2 was used as a loading control (lower panels). The OD of the pY-STAT3 band in untreated, IL-6-stimulated cells was arbitrarily given the value of 1.0. The numbers given under the different panels [relative units (Rel. Units)] represent the relative intensities of the protein bands from two independent experiments. The positions of pY-STAT3 and ERK1/2 are indicated on the left and that of the molecular mass markers (in kDa) are shown on the right. C, PDTC decreases nuclear translocation of STAT3 in HepG2 cells. Cells were preincubated with vehicle (veh.) or 50 μm PDTC for 2 h, then treated with 20 ng/ml IL-6 for 20 min. Cells were fixed and probed with STAT3 antibody to detect subcellular localization of STAT3. Nuclei were visualized by staining with Topro. Confocal images in the bottom panels are overlay of STAT3 and Topro images. The results are representative to three independent experiments. D, Effect of PDTC on FBG protein levels of HepG2 cells stimulated with IL-6. HepG2 cells were left untreated or treated with PDTC for 2 h before the addition of IL-6 (20 ng/ml) or IL-1β (20 ng/ml) for 24 h. Cells were lysed and analyzed by immunoblotting for expression of FBG (top panel), pY-STAT3 (middle panel), and ERK1/2 (lower panel) as a loading control. Similar results were obtained in two independent experiments.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Incubation, Western Blot, Translocation Assay, Staining, Expressing

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: STAT3 contains cysteine residues that are targets for oxidant-mediated S-glutathionylation. A and B, Serum-starved HepG2 cells were left untreated or incubated either with PDTC (50 μm, 2 h), GSH disulfide (GSSG, 20 mm, 30 min) or diamide (500 μm, 30 min), after which cells were homogeneized in RIPA buffer supplemented with the thiol biotinylating agent, MBB (200 μm), for 30 min on ice. A, After quenching the alkylation reaction with excess l-cysteine, the clarified cell lysates were subjected to immunoprecipitation with STAT3 antibody and immunoblotted with streptavidin-conjugated HRP (top panel) and STAT3 (lower panel) as a loading control. B, Alternatively, the clarified cell lysates were incubated with CaptAvidin-linked agarose for 1 h at 4 C, and the immobilized proteins were eluted with biotin, followed by Western blot analysis with anti-STAT3. The band intensity of thiol-biotinylated STAT3 in untreated cells was arbitrarily given the value of 1.0. Results are representative of two separate experiments with similar results. C, Reversible oxidation of STAT3 cysteine residues by PDTC. HepG2 cells were treated without or with PDTC for 2 h. Cell lysates were subjected to biotin switch assay, followed by affinity precipitation with streptavidin-agarose and then immunoblot analysis with anti-STAT3 antibody. Bands were detected only in PDTC-treated cells and when biotin-HPDP was included in the assay. D, HepG2 cells were left untreated or incubated with PDTC, GSSG, or diamide as indicated in A. STAT3 immunoprecipitates were separated by SDS-PAGE under nonreducing conditions, and analyzed by Western blot with a monoclonal antibody against protein-bound GSH (αSSG, top panel) and STAT3 (lower panel) as a loading control. The S-glutathionylated STAT3 band intensity in untreated cells was arbitrarily given the value of 1.0. Rel. Units, Relative units; veh, vehicle.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Incubation, Immunoprecipitation, Western Blot, Biotin Switch Assay, Affinity Precipitation, SDS Page

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: In vitro S-glutathionylation inhibits JAK2-mediated STAT3 phosphorylation and its DNA-binding activity. A, STAT3 is an in vitro target of glutathionylation/deglutathionylation reaction. Anti-STAT3 immunoprecipitates from control HepG2 cells were left untreated (lane 1) or treated with diamide (1 mm)/2 mm GSH for 1 h at room temperature (lanes 2–5), after which vehicle (veh.), recombinant E. coli GRX-1 or DTT was added for 30 min. The immune pellets were resolved by SDS-PAGE under nonreducing conditions, and analyzed by Western blot with antibodies raised against αSSG (top panel) and STAT3 as a loading control (bottom panel). B, STAT3 immunoprecipitates were left untreated or treated with 1 mm diamide/GSH as described under Materials and Methods. Tyrosine phosphorylation was then performed using recombinant active JAK2 for 10 min at 30 C. The proteins in the immune pellets were analyzed by Western blot using antibodies against pY-STAT3 (upper panel) and total STAT3 (lower panel). The reversibility of the inhibitory effect of diamide was shown by using 2 mm DTT before the JAK2-mediated phosphorylation step. The numbers given under the panel [relative units (Rel. Units)] represent the quantitated ratios of pY-STAT3 to total STAT3 relative to that of untreated vehicle control. Results are representative of two independent experiments. C, Cells were treated in the absence or the presence of IL-6 (20 ng/ml) for 30 min. Nuclear extracts were prepared, and DNA binding activity of STAT3 was determined as described under Materials and Methods. Nuclear extracts were incubated with agarose-bound oligonucleotide probe containing the wild-type (wt) GAS consensus sequence (lanes 1–2) or the same probe mutated (mut) at the GAS site (lanes 3–4). Bound STAT3 was resolved by SDS-PAGE, and analyzed by Western blot with anti-pY-STAT3. An identical result was obtained in an additional experiment. D, The nuclear extracts of IL-6-stimulated cells were treated without or with 3 mm GSSG for 1 h at 37 C to induce in vitro S-glutathionylation. STAT3 immobilized with agarose-bound wild-type oligonucleotide probe was analyzed by Western blot using anti-pY-STAT3. The numbers given in the panel (Rel. Units) represent the intensities of pY-STAT3 protein bands relative to that of control IL-6-stimulated cells. Results are representative of four independent observations from two separate experiments with similar results.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: In Vitro, Binding Assay, Activity Assay, Recombinant, SDS Page, Western Blot, Incubation, Sequencing

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: Ectopic expression of GRX-1 enhances IL-6-induced STAT3 signaling. Cells were transiently transfected with Flag-STAT3 together with pcDNA vector or GRX-1 plasmid. A, Forty-eight hours later, cells were homogeneized in RIPA buffer containing MBB, and anti-Flag immunoprecipitates were analyzed by Western blot using HRP-conjugated streptavidin (Strep.) (top panel) or anti-STAT3 (second panel) and anti-GRX-1 (bottom panel) antibodies. B, Serum-starved transfected cells were left untreated or incubated with diamide (500 μm) for 30 min, followed by the addition of IL-6 (20 ng/ml) for 20 min. The anti-Flag immunoprecipitates were analyzed by Western blotting for the detection of pY-STAT3 (top panel), and Flag-STAT3 (second panel). Immunoblot analysis showed detection of GRX only in clarified lysates of GRX-1-transfected cells (third panel). Endogenous ERK 1/2 was detected at comparable levels in each lane (bottom panel). The pY-STAT3 band intensity in Flag-STAT3-transfected cells stimulated with IL-6 was arbitrarily given the value of 1.0. No pY-STAT3 signal was detected in the absence of IL-6 (data not shown). IP, Immunoprecipitation; IB, immunoblots. C, The levels of Flag-STAT3 in the nuclear (Nuc) (second panel) and cytosolic (Cyt) (third panel) fractions of serum-starved cells exposed to IL-6 (20 ng/ml) for 30 min were assessed by Western blot analysis. The membranes were reprobed with BRG-1 (top panel) and ERK 1/2 (bottom panel) antibodies to demonstrate equal protein loading in each lane. D, Nuclear Flag-STAT3 band intensities were normalized to cellular pool (nuclear + cytosolic) of Flag-STAT3 protein. The dot plot represents the individual measurements that were derived from two separate experiments, each performed in duplicate dishes. Rel. Units, Relative units; veh., vehicle.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Incubation, Immunoprecipitation, Derivative Assay

Journal:

Article Title: S -Glutathionylation Impairs Signal Transducer and Activator of Transcription 3 Activation and Signaling

doi: 10.1210/en.2008-1241

Figure Lengend Snippet: Effect of GRX-1 overexpression on STAT3 DNA binding and induction of target genes. A, ChIP assays using agarose-conjugated Flag antibody (M2-Ag) were performed in HepG2 cells transfected with Flag-STAT3 plasmid together with empty vector (pcDNA) or GRX-1 plasmid, and then left untreated or stimulated with IL-6 for 30 min. The results are expressed as percentage of immunoprecipitated (IP) DNA to total DNA input (input). Similar results were obtained in three independent experiments. B, HepG2 cells were transiently cotransfected with pGAS-TA-Luc reporter plasmid, pCMVSport-β-galactosidase expression plasmid, and Flag-tagged STAT3 together with either pcDNA empty vector or GRX-1 plasmid. Twenty-four hours later, the cells were serum starved for 4 h and then pretreated with vehicle (veh.) (open bars) or 20 ng/ml IL-6 (filled bars) for 6 h. Cell extracts were analyzed for luciferase activity and normalized for β-galactosidase. All values are expressed relative to that of Flag-STAT3-expressing cells without IL-6. Results are expressed as means ± sd of a single experiment performed in triplicate dishes. Results are representative of two separate experiments with similar results.

Article Snippet: Full-length cDNA encoding human STAT3 (pCMV6-XL4; OriGene Technologies, Inc., Rockville, MD) was subcloned in-frame into N-terminal p3xFLAG-CMV (Sigma-Aldrich Corp., St. Louis, MO) to generate the Flag-STAT3 fusion construct.

Techniques: Over Expression, Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Luciferase, Activity Assay

Journal: Cell Death & Disease

Article Title: Axotomy induces axonogenesis in hippocampal neurons through STAT3

doi: 10.1038/cddis.2011.59

Figure Lengend Snippet: STAT3 is required for axotomy-induced axonogenesis. ( a ) Western blot analysis for the detection of STAT3. The hippocampal neurons at 3DIV were cotransfected with STAT3 siRNA or control siRNA in combination with GFP. ( b ) Immunostaining for STAT3 shows that the expression of STAT3 decreased in the STAT3 siRNA-transfected neurons compared to non-transfected neighboring neurons. Scale bar, 50 μ m. ( c ) Number of neurites. Cultured hippocampal neurons were transfected with STAT3 siRNA ( n =23) or control siRNA ( n =20). Data are represented as mean±S.E.M. ( d ) Axonal length. Data are represented as mean±S.E.M. ( e ) Number of neurons with the indicated morphological changes. No axonogenesis occurred after axotomy in the neurons transfected with STAT3 siRNA ( n =38). Control siRNA, n =17. ( f ) Number of neurons with and without axonogenesis. Knockdown of STAT3 inhibited axonogenesis. ** P <0.01

Article Snippet: Cells were then resuspended in 100 μ l of rat neuron Nucleofector solution (Amaxa Biosystems) at RT, and 5 μ g of plasmids were added, including GFP-expression vector (Amaxa Biosystems) and Flag-STAT3-C Flag pRc/CMV (Addgene, Cambridge, MA, USA) at the ratio of 1 : 10.

Techniques: Western Blot, Immunostaining, Expressing, Transfection, Cell Culture

Journal: Cell Death & Disease

Article Title: Axotomy induces axonogenesis in hippocampal neurons through STAT3

doi: 10.1038/cddis.2011.59

Figure Lengend Snippet: Overexpression of STAT3 enhances axotomy-induced axonogenesis. ( a ) Cultured hippocampal neurons at 3 DIV were transfected with Flag-STAT3 and GFP. Scale bar, 50 μ m. ( b ) Number of neurites. Cultured hippocampal neurons were transfected with GFP ( n =25) or GFP plus Flag-STAT3 ( n =28). Data are represented as mean±S.E.M. ( c ) Axonal length. Data are represented as mean±S.E.M. ( d and e ) Axonogenesis after axotomy was increased in the neurons expressing GFP plus Flag-STAT3 ( n =28). GFP, n =52. * P <0.05

Article Snippet: Cells were then resuspended in 100 μ l of rat neuron Nucleofector solution (Amaxa Biosystems) at RT, and 5 μ g of plasmids were added, including GFP-expression vector (Amaxa Biosystems) and Flag-STAT3-C Flag pRc/CMV (Addgene, Cambridge, MA, USA) at the ratio of 1 : 10.

Techniques: Over Expression, Cell Culture, Transfection, Expressing