expression vector human stat3  (Servicebio Inc)

Journal: Frontiers in Pharmacology

Article Title: Lycorine Displays Potent Antitumor Efficacy in Colon Carcinoma by Targeting STAT3

doi: 10.3389/fphar.2018.00881

Figure Lengend Snippet: Lycorine targets STAT3 in CRC Cells. (A,B) cells were treated with 40 μM of lycorine for 24 h and subsequently heated at different temperature for 3 min. after freeze-thaw cycles for cell lysis, the soluble STAT3 protein levels bound to a drug were visualized by Western blot assays. (C) Pull-down assay showing an interaction between lycorine and STAT3. Lycorine was conjugated with epoxy-activated Sepharose 6B. (D–F) Docking model of lycorine with STAT3. (D) The interaction pattern of lycorine with the residues. (E) Lycorine binding with the pocket is composed of hydrogen bonds. (F) 2D diagram between the receptor and ligand. All the western data shown are representative of at least three independent experiments.

Article Snippet: Expression vector of human STAT3 was designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Lysis, Western Blot, Pull Down Assay, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Lycorine Displays Potent Antitumor Efficacy in Colon Carcinoma by Targeting STAT3

doi: 10.3389/fphar.2018.00881

Figure Lengend Snippet: Lycorine promotes caspase-dependent mitochondrial apoptosis via STAT3 inactivation. (A , B) RKO and SW480 cells were treated with indicated concentrations of lycorine for 48 h. The protein levels of p-STAT3 were determined by Western blot assays. Total STAT3 expressions were detected as the internal control. (C–E) Cells transfected with STAT3 (STAT3 Vec) or empty vector (Control Vec) followed by lycorine treatment. (C) The protein levels of p-STAT3, STAT3, PARP, cleaved caspase-3, Bax, and Bcl-2 were detected by Western blot assays. (D) The colony formation capability was detected by colonogenic assay. (E) The cell viability was measured by MTT assay. For (E,F) , data are shown as mean ± SD ( n = 3); ∗∗ P < 0.01 compared with Vector control; ## P < 0.01 compared with Vector control transfected cells treated with lycorine (Student’s t -test). All the western data shown are representative of at least three independent experiments.

Article Snippet: Expression vector of human STAT3 was designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, MTT Assay

Journal: Frontiers in Pharmacology

Article Title: Lycorine Displays Potent Antitumor Efficacy in Colon Carcinoma by Targeting STAT3

doi: 10.3389/fphar.2018.00881

Figure Lengend Snippet: Lycorine blocks growth and development in colorectal SW480 xenograft tumors. (A–H) BALB/c nude mice were inoculated with SW480 cells and treated with lycorine or vehicle. (A) Tumors were isolated and photographed. (B) Tumors were weighted. (C) Bodies were weighted. (D) Tumor volumes were measured every 3 days. (E) PARP, cleaved caspase-3 and Bcl-2 levels were determined in xenograft tumors by Western blot assays. (F) PARP and cleaved caspase-3 expressions were determined by IHC staining in xenograft tumors. (G) p-STAT3 and total STAT3 levels were determined in xenograft tumors by Western blot assays. (H) p-STAT3 expressions were determined by IHC staining in xenograft tumors. For (B , D) , data are shown as mean ± SD ( n = 9). ∗ P < 0.05; ∗∗ P < 0.01 compared with control (Student’s t -test). For (F , H) , representative images were conducted as indicated. ∗∗∗ P < 0.001; Scale bars: 50 μm. All the western data shown are representative of at least three independent experiments.

Article Snippet: Expression vector of human STAT3 was designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Isolation, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Magnolin promotes autophagy and cell cycle arrest via blocking LIF/Stat3/Mcl-1 axis in human colorectal cancers

doi: 10.1038/s41419-018-0660-4

Figure Lengend Snippet: a HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. The protein levels of p-Stat3 were determined by western blot assays. Total Stat3 expressions were detected as the internal control. b Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec). The levels of LIF, p-Stat3, and Total Stat3 proteins were detected by western blot assays. c Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. The levels of p-Stat3 and Total Stat3 proteins were determined by western blot assays. d – j Cells were transfected with Stat3 (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. d , e The protein levels of Stat3 and Mcl-1 were determined by western blot assays. The mRNA levels of Mcl-1 were detected by real-time PCR. f The protein levels of LC-3B, p62, Cyclin D1, and p27 were detected by western blot assays. g , h The cell cycle distribution was determined by flow cytometer. i , j Cells were transfected with a reporter plasmid (mRFP-GFP-LC3), followed by a confocal laser scanning microscope. For ( d , e ) and ( g – j ), data are shown as mean ± s.d. ( n = 3); ** P < 0.01 compared with vector control transfected cells; ## P < 0.01 compared with vector control transfected cells treated with magnolin (Student’s t test). All the western data shown are representative of at least three independent experiments

Article Snippet: Expression vectors of human LIF, Stat3, and Mcl-1 were designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Flow Cytometry, Laser-Scanning Microscopy

Journal: Cell Death & Disease

Article Title: Magnolin promotes autophagy and cell cycle arrest via blocking LIF/Stat3/Mcl-1 axis in human colorectal cancers

doi: 10.1038/s41419-018-0660-4

Figure Lengend Snippet: a – g BALB/c nude mice were inoculated with HCT116 cells and treated with magnolin or vehicle. a Tumors were isolated and photographed. b Tumors were weighted. c Tumor volumes were measured every 3 days. d Bodies were weighted. e , f LIF, p-Stat3, and Mcl-1 expressions were determined by IHC staining in xenograft tumors. Representative images were conducted as indicated. *** P < 0.001; Scale bars, 50 μm. g LIF, p-Stat3, Stat3, and Mcl-1 levels were determined in xenograft tumors by western blot assays. Western data shown are representative of at least three independent experiments. h Kaplan–Meier plots of the relapse-free survival (RFS) of CRC patients, stratified by expression of LIF or Mcl-1. Data obtained from the publically available datasets (GSE17536). i The relevance between LIF and relative expression of Mcl-1 in clinical CRC samples. j Representative micrographs of LIF, p-Stat3, and Mcl-1 expression in CRC and normal colon tissues, as analyzed by IHC. Scale bars, 200 μm. k The overall survival of CRC patients with LIF and Mcl-1 expression, as well as combined expression of LIF and Mcl-1 ( l ) are analyzed by the Kaplan–Meier estimates and the log-rank test. For ( a – g ), data are shown as mean ± s.d. ( n = 9). * P < 0.05; ** P < 0.01 compared with control (Student’s t test)

Article Snippet: Expression vectors of human LIF, Stat3, and Mcl-1 were designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Isolation, Immunohistochemistry, Western Blot, Expressing, Control

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