Journal:

Article Title: Regulation of Id Gene Expression by Type I Insulin-Like Growth Factor: Roles of STAT3 and the Tyrosine 950 Residue of the Receptor

doi: 10.1128/MCB.21.16.5447-5458.2001

Figure Lengend Snippet: IGF-I is required for up-regulation of Id2 RNA and inhibition of differentiation by DNStat3. (A) Growth of 32D IGF-IR/DN Stat3 cells was determined in 10% serum in the presence or absence of IGF-I (50 ng/ml). The results given were obtained at 96 h after IL-3 withdrawal. (B) Expression of Id2 mRNA in parental 32D cells, 32D IGF-IR cells, and 32D IGF-IR/DNStat3. The cells were exponentially growing either in IL-3 or in IGF-I, as indicated above the lanes. IGF-I was added for 6 h. (C) Id2 mRNA levels in mouse embryo fibroblasts (R600 cells). The cells were either quiescent (lane 2) or stimulated (lane 1). RNA amounts in the last two panels were monitored with rRNA.

Article Snippet: After being blotted, the membrane was probed with a polyclonal antibody against antiphospho-Stat3 Y705 (New England Biolabs), stripped, and reprobed with a Stat3 monoclonal antibody (Transduction Lab.).

Techniques: Inhibition, Expressing

Journal:

Article Title: Regulation of Id Gene Expression by Type I Insulin-Like Growth Factor: Roles of STAT3 and the Tyrosine 950 Residue of the Receptor

doi: 10.1128/MCB.21.16.5447-5458.2001

Figure Lengend Snippet: Phosphorylation of tyrosine 705 of Stat3 by IGF-I. The cell lines are indicated above the panels. (A) The times (in minutes) are times after placing the cells in serum-free medium (SFM) and stimulation with IGF-I. The lysates were immunoprecipitated with an antibody to Stat3, and the blots were developed with a phosphoantibody to tyrosine 705 of Stat3. The blots were then reprobed with an antibody to Stat3. (B) Western blots on lysates of the same cell lines. The blots were probed directly with an antibody to Y705 (upper row) and then reprobed with anti-Stat3 antibody (lower row). In the experiment on the right, G-CSF was used as control for the phosphorylation of Y705. (C) A mutation at Y950 of the IGF-IR abrogates the detection of Y705 phosphorylation of Stat3, regardless of the presence or absence of DNStat3 (left). The right panel shows that Y705 phosphorylation of Stat3 is not abrogated in the same cell lines stimulated with G-CSF.

Article Snippet: After being blotted, the membrane was probed with a polyclonal antibody against antiphospho-Stat3 Y705 (New England Biolabs), stripped, and reprobed with a Stat3 monoclonal antibody (Transduction Lab.).

Techniques: Immunoprecipitation, Western Blot, Mutagenesis

Journal:

Article Title: Regulation of Id Gene Expression by Type I Insulin-Like Growth Factor: Roles of STAT3 and the Tyrosine 950 Residue of the Receptor

doi: 10.1128/MCB.21.16.5447-5458.2001

Figure Lengend Snippet: A dominant negative mutant of Stat3 inhibits IGF-I-mediated differentiation and causes transformation of 32D IGF-IR cells. 32D cells were transduced with the appropriate retroviral vectors, and mixed populations were selected. (A) IGF-IR cells and 32D IGF-IR/DN Stat3 cells were grown in medium supplemented with 10% serum and either IL-3 or IGF-I. The percentage of differentiated cells is indicated on the ordinate. In this experiment, the cells were fixed and stained after 4 days in the indicated medium. The percentage of differentiated cells was determined by standard methods (60). (B) After withdrawal of IL-3, the cells were grown in medium supplemented with 10% serum and either IGF-I or insulin at a concentration of 50 ng/ml. The cells were counted 48 h after IL-3 withdrawal. The cell lines are indicated on the left of the figure. 32D, parental cells; 32D IR, cells overexpressing the insulin receptor; 32D IGF-IR, cells expressing increased levels of IGF-IR. DN/STAT3 indicates the same cell lines stably transduced with the dominant negative mutant of Stat3. The inset shows levels of expression of DN/Stat3, after immunoprecipitation with a FLAG antibody and blotting with an anti-Stat3 antibody. Lanes of inset: 1, 32D/DN Stat3; 2, 32D IR/DN Stat3; 3, 32D IGF-IR/DN Stat3; 4, parental 32D cells.

Article Snippet: After being blotted, the membrane was probed with a polyclonal antibody against antiphospho-Stat3 Y705 (New England Biolabs), stripped, and reprobed with a Stat3 monoclonal antibody (Transduction Lab.).

Techniques: Dominant Negative Mutation, Transformation Assay, Transduction, Staining, Concentration Assay, Expressing, Stable Transfection, Immunoprecipitation

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Targeting STAT3 restores BRAF inhibitor sensitivity through miR-759-3p in human cutaneous melanoma cells

doi:

Figure Lengend Snippet: The STAT3 activity confers BRAF inhibitor resistance in BRAF mutant melanoma cells. A. Proliferation of A375-S, WM35-S and the vemurafenib-resistant A375-R and WM35-R cells in the presence of PLX4032 (PLX) for 24 hs. IC50 values are means (μM; n = 3) ± standard error of mean. B. Western blot for phosphorylated STAT3 and STAT3 in A375-S, WM35-S, A375-R and WM35-R cells. C. Phosphorylated STAT3 were analyzed by immunofluorescence staining and confocal microscopy.

Article Snippet: Antibodies against pSTAT3 (Y705), BRAF, STAT3 and actin were obtained from Santa Cruz Biotechnology.

Techniques: Activity Assay, Mutagenesis, Western Blot, Immunofluorescence, Staining, Confocal Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150, decreases neuroblastoma tumorigenicity and increases chemosensitivity

doi: 10.1158/1078-0432.CCR-16-1317

Figure Lengend Snippet: A. STAT3 mRNA expression in neuroblastoma cells expressing tetracycline-inducible STAT3 shRNA was validated by quantitative PCR (qPCR). Cells were treated with Tet (1 µg/ml) or solvent control 1, 3, and 6 days prior to harvesting. The data is presented as the mean of 3 replicate tests ± SE. *, P<0.05 was indicated for tetracycline treated cells vs. control cells (t-test).

Article Snippet: Antibodies against phosphorylated STAT3 (Y705, S727), STAT3, CyclinD1, CyclinD3, Bcl-2, Survivin and GAPDH were purchased from Santa Cruz (Santa Cruz, CA).

Techniques: Expressing, shRNA, Real-time Polymerase Chain Reaction

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150, decreases neuroblastoma tumorigenicity and increases chemosensitivity

doi: 10.1158/1078-0432.CCR-16-1317

Figure Lengend Snippet: A. STAT3 mRNA expression in neuroblastoma cell lines treated with AZD9150 (0.25, 0.5, 1, 2.5, 5, 10 µM) or ntASO (10 µM) for 6 days was validated by qPCR. The data is presented as the mean of 3 replicate tests ± SE. *, P<0.05 was indicated for AZD9150 treated cells vs. control cells (t-test).

Article Snippet: Antibodies against phosphorylated STAT3 (Y705, S727), STAT3, CyclinD1, CyclinD3, Bcl-2, Survivin and GAPDH were purchased from Santa Cruz (Santa Cruz, CA).

Techniques: Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150, decreases neuroblastoma tumorigenicity and increases chemosensitivity

doi: 10.1158/1078-0432.CCR-16-1317

Figure Lengend Snippet: A. The growth of neuroblastoma cells expressing Tet-inducible STAT3 shRNA with Tet (1µg/ml) or solvent control was measured using IncuCyte (Essen BioSciences, USA) every 6h. P-value between solvent control and Tet-treated group was determined by a two-way ANOVA.

Article Snippet: Antibodies against phosphorylated STAT3 (Y705, S727), STAT3, CyclinD1, CyclinD3, Bcl-2, Survivin and GAPDH were purchased from Santa Cruz (Santa Cruz, CA).

Techniques: Expressing, shRNA

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150, decreases neuroblastoma tumorigenicity and increases chemosensitivity

doi: 10.1158/1078-0432.CCR-16-1317

Figure Lengend Snippet: A. Subcutaneous xenografts of NB were established by injection of 2 × 106 cells of NGP expressing Tet-inducible STAT3 shRNA into the right flank of 5–6 week-old female nude mice. Mice were given water supplemented with food containing doxycycline (Tet+) or regular food (Tet−) 1 week before tumor implantation and continued this way throughout the experiment. The graph represents a comparison of mean tumor volumes between normal food (Tet−) or doxycyline food treatment groups (Tet+). The days were counted when the size of the tumors reached 100–200 mm3. Data represents mean ± SE of n=7.

Article Snippet: Antibodies against phosphorylated STAT3 (Y705, S727), STAT3, CyclinD1, CyclinD3, Bcl-2, Survivin and GAPDH were purchased from Santa Cruz (Santa Cruz, CA).

Techniques: Injection, Expressing, shRNA, Tumor Implantation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibition of STAT3 with the generation 2.5 antisense oligonucleotide, AZD9150, decreases neuroblastoma tumorigenicity and increases chemosensitivity

doi: 10.1158/1078-0432.CCR-16-1317

Figure Lengend Snippet: A. Subcutaneous xenografts of NB were established by injection of 2×106 NGP expressing Tet-inducible STAT3 shRNA cells into the right flank of 5–6 week-old female nude mice. Mice were given water supplemented with food containing doxycycline (Tet+) or regular food (Tet−) 1 week before tumor implantation which was continued throughout the experiment. Cisplatin treatment (2 mg/kg) was initiated when tumors reached 100–200 mm3. Cisplatin (2 mg/kg) was injected twice a week intraperitoneally. The tumor size in treated groups was compared to that of Tet− group. Data represents mean ± SE of n=7 mice per cohort. P value was calculated using student t-test. *, P<0.05 was indicated for the combination of Tet+ and cisplatin treated mouse vs. Tet− mouse.

Article Snippet: Antibodies against phosphorylated STAT3 (Y705, S727), STAT3, CyclinD1, CyclinD3, Bcl-2, Survivin and GAPDH were purchased from Santa Cruz (Santa Cruz, CA).

Techniques: Injection, Expressing, shRNA, Tumor Implantation

Journal: Oncogene

Article Title: EZH2-mediated upregulation of ROS1 oncogene promotes oral cancer metastasis

doi: 10.1038/onc.2017.262

Figure Lengend Snippet: Effect of increased ROS1 on MAPK, PI3K-AKT signaling pathways, cell proliferation, migration, and invasion. ( a ) Western blotting was carried out with lysates of OC3 and OC3-IV2 cells using anti-ROS1 and anti-pROS1(Y2274). The value for pROS1(Y2274) was normalized to total ROS1. ( b and c ) Relative protein levels of pAKT(S473), AKT, pERK1/2, ERK1/2, pSTAT3(Y705), and STAT3 in OC3, OC3-IV2, OC3-IV2-Scr, and OC3-IV2-shROS1#1 cells as determined with Western blotting. Levels of pERK1/2, pAKT(S473), and pSTAT3(Y705) were normalized to those of total ERK1/2, AKT, and STAT3, respectively, and then to OC3 and OC3-IV2-Scr cells. ( d ) pAKT(S473), AKT, pERK1/2, and ERK1/2 levels in lysates from OC3 and OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 24 h. ( e and f ) Relative proliferation and migration of OC3 and OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 24 or 48 h were assessed using MTT and wound-healing assays, respectively. * , # Compared with DMSO treatment. ( g ) Migration and invasion of OC3-IV2 cells treated with 20 μ M U0126 or LY294002 for 26 h were assessed with the Boyden chamber assay. Data from at least three independent experiments are presented as mean±SEM (* , # P <0.05).

Article Snippet: Antibody against pSTAT3(Y705) (#BS4181) was purchased from Bioworld Technology (Louis Park, MN, USA).

Techniques: Migration, Western Blot, Boyden Chamber Assay

Journal: Frontiers in Immunology

Article Title: Epstein Barr Virus Interleukin 10 Suppresses Anti-inflammatory Phenotype in Human Monocytes

doi: 10.3389/fimmu.2018.02198

Figure Lengend Snippet: vIL-10 induces limited STAT3 phosphorylation in monocytes. Monocytes were enriched from PBMCs using magnetic beads and stimulated with 10 ng/ml hIL-10 or vIL-10 for 30 min. pSTAT3 (pY705) was measured by flow cytometry. (A) Representative histogram for pSTAT3. (B) Mean fluorescence intensities for pSTAT3 are shown as fold increase over unstimulated. Data are presented as mean ± SD, n = 13, *** p < 0.001 by Wilcoxon test. (C) Western blot analysis shows increased STAT3 phosphorylation in cells stimulated with hIL-10 compared to those stimulated with vIL-10. Cells from three independent donors were used. Upper panel: phosphorylated-STAT3, lower panel: total STAT3. (D) pSTAT3 presented as ratio of band intensity of pSTAT3 and STAT3 from (C) for each donor. * p < 0.05, ** p < 0.01 by ANOVA.

Article Snippet: STAT3 phosphorylation was detected by flow cytometry using antibodies directed against phospho-STAT3 Y705 (BD Biosciences).

Techniques: Magnetic Beads, Flow Cytometry, Fluorescence, Western Blot

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: Comparison between MCF10A and H- Ras MCF10A cells and the effect of curcumin on induction of apoptosis. ( A ) Total cell lysates of MCF10A and H- Ras MCF10A cells were prepared, and the expression of phosphorylated STAT3 was assessed by Western blot analysis. ( B ) The same number of MCF10A and H- Ras MCF10A cells (2 × 10 3 ) was seeded onto Lipidure®-Coat Plate A-U96 and incubated for 5 days. The spheroid formations were observed under the microscope. scale bar, 500 μm. Data are means ± standard deviation (SD). ns: not significant; * P < 0.05; ** P < 0.005; *** P < 0.001. ( C ) The MTT assay was conducted with MCF10A and H- Ras MCF10A cells treated with various concentrations of curcumin for indicated time. Data are means ± SD (* P < 0.05). ( D ) H- Ras MCF10A cells were treated for 24 h with indicated concentrations of curcumin. The expression of cleaved PARP, Bcl-2 and cleaved caspase 3 was observed by Western blot analysis. ( E ) H- Ras MCF10A cells were treated with curcumin (10 μM, 25 μM or 50 μM) for 24 h and then incubated with Annexin V and PI for 15 min. The proportion of apoptotic cells was measured by FACS analysis.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Comparison, Expressing, Western Blot, Incubation, Microscopy, Standard Deviation, MTT Assay

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: Curcumin-induced apoptosis of H- Ras MCF10A cells through inhibition of STAT3 signaling. ( A ) H- Ras MCF10A cells were treated with indicated concentrations of curcumin for 24 h. The expression of phosphorylated STAT3 was assessed by Western blot analysis. Actin was used as an equal loading control. * P < 0.05; *** P < 0.001. ( B ) Luciferase activity was measured with HeLa/P-STAT3-luc cells preincubated with indicated concetrations of curcumin for 12 h and then stimulated with OSM for 6 h. * P < 0.05; ** P < 0.005; *** P < 0.001. ( C ) H- Ras MCF10A cells were incubated with curcumin (25 μM) for indicated time periods. The expression of phosphorylated STAT3 and upsteam kinases was determined by Western blot analysis. ( D ) H- Ras MCF10A cells were treated with scrambled or STAT3 siRNA for 48 h. PARP cleavage and Bcl-2 expression were detected by Western blot analysis.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Inhibition, Expressing, Western Blot, Control, Luciferase, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: Involvement of the α,β unsaturated carbonyl group of curcumin in its inhibition of STAT3 activation and induction of apoptosis. ( A ) Curcumin has the two α, β unsaturated carbonyl groups which are eliminated in tetrahydrocurcumin by hydrogenating the double bonds. ( B ) H- Ras MCF10A cells were incubated with vehicle, curcumin (25 μM) or tetrahydrocurcumin (25 μM). The expression of phosphorylated STAT3, STAT3 and related kinase was determind by Western blot analysis. ( C ) H- Ras MCF10A cells were treated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h and then incubated with Annexin V and PI for 15 min. The population of apoptotic cells was measured by FACS analysis. ( D ) H- Ras MCF10A cells were cultured in soft agar for 10 days. Curcumin or tetrahydrocurcumin was added to the medium every other day for another 10 days. The colony formation was visualized under the microscope. ** P < 0.005. scale bar, 100 µm.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Inhibition, Activation Assay, Incubation, Expressing, Western Blot, Cell Culture, Microscopy

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: The comparative effect of curcumin and tetrahydrocurcumin on STAT3 dimerization. ( A ) Total lysate from H- Ras MCF10A cells treated with curcumin (25 μM) for 12 h was immunoprecipitated with Protein A/G agarose and anti-STAT3 antibody overnight and analyzed by immunobloting with anti-STAT3 antibody. * P < 0.05; ** P < 0.005. ( B ) PC-3 cells were co-transfected with HA-tagged STAT3 and Myc-tagged STAT3 and treated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h. The total lysates obtained from the transfected cells were immunoprecipitated with anti-Myc antibody and analyzed by Western blotting with anti-HA antibody. * P < 0.05; ** P < 0.005.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Immunoprecipitation, Western Blot, Transfection

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: The effect of curcumin and tetrahydrocurcumin on phosphorylation, nuclear translocation, and transcriptional activity of STAT3. ( A ) H- Ras MCF10A cells were treated with curcumin and tetrahydrocurcumin (25 μM each) for 12 h. Cytosolic and nuclear fractions were extracted and subjected to Western blot analysis for measuring total and phosphorylated STAT3 levels. ( B ) Luciferase activity was measured in HeLa/P-STAT3-luc cells preincubated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h and then stimulated with OSM for additional 6 h. * P < 0.05; ** P < 0.005; *** P < 0.001. ( C ) A chromatin immunoprecipitation (ChIP) assay was conducted using chromatin prepared from H- Ras MCF10A cells treated with curcumin or tetrahydrocurcumin (25 μM each) for 12 h. The binding of STAT3 at the promoter of its target genes ( Survivin , Bcl-2 and Bcl-xL ) was determined. ( D ) H- Ras MCF10A cells were treated with curcumin or tetrahydrocurcumin (25 μM each) for 8 h or 12 h. The expression of Bcl-2, Bcl-xL and Survivin was analyzed by Western blot analysis.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Translocation Assay, Activity Assay, Western Blot, Luciferase, Chromatin Immunoprecipitation, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: STAT3 thiol modification by curcumin. ( A ) H- Ras MCF10A cells were treated with curcumin (25 μM) in the presence or absence of NAC (10 mM) or DTT (0.5 mM). ( B ) Luciferase activity was measured with HeLa/P-STAT3-luc cells preincubated with curcumin (25 µM) in the presence or absence of NAC (10 mM) or DTT (0.5 mM) for 12 h and then stimulated with OSM for another 6 h. ** P < 0.005; *** P < 0.001. ( C ) Covalent binding of curcumin to STAT3 as predicted by computational modeling. AutoDock Vina program and GOLD5.0 docking program were used in docking experiments for cysteine 251 and 259, respectively.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Modification, Luciferase, Activity Assay, Binding Assay

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: Identification of curcumin binding sites in recombinant STAT3 by LC-MS/MS. Annotated mass spectrum illustrating the binding of curcumin to STAT3 at cysteine 251 and 259 residues was observed. MS/MS spectrum of precursor ion at m/z 1033.49304 [M + 2 H] +2 corresponds to the amino acid sequence of STAT3. The elucidation of fragment ion at m/z 177.05501 from the structure was given in the inset of the spectra. The sample preparation and other experimental details for mass spectral analysis are described in Methods.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Binding Assay, Recombinant, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing, Sample Prep

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: Direct interaction of curcumin with endogenous STAT3 and the cysteine 259 residue of STAT3 as a putative binding site. ( A ) Curcumin-conjugated Sepharose-4B and tetrahydrocurcumin-conjugated Sepharose-4B were incubated with H- Ras MCF10A cell lysates overnight. The STAT3 bound to the curcumin- and tetrahydrocurcumin-conjugated Sepharose 4B beads was pulled down by centrifugation and detected by Western blot analysis. ( B ) PC-3 cells transiently transfected with GFP-tagged WT STAT3, GFP-tagged C251A or GFP-tagged C259A were stimulated with IL-60 (50 ng/ml) for 12 h. The cell lysates were incubated with curcumin-conjugated Sepharose-4B. WT STAT3 and STAT3 mutants were detected by anti-STAT3 antibody. * P < 0.05; ** P < 0.005; *** P < 0.001. ( C ) PC3 cells were transiently transfected with GFP-tagged WT STAT3, or GFP-tagged mutant STAT3 (STAT-C251A or C259A) and then treated with IL-6 (50 ng/ml) for 12 h, followed by curcumin treatment (25 µM) for 12 h. The expression of cleaved PARP was determined by Western blot analysis. * P < 0.05; ** P < 0.005; *** P < 0.001.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Residue, Binding Assay, Incubation, Centrifugation, Western Blot, Transfection, Mutagenesis, Expressing

Journal: Scientific Reports

Article Title: Curcumin interacts directly with the Cysteine 259 residue of STAT3 and induces apoptosis in H- Ras transformed human mammary epithelial cells

doi: 10.1038/s41598-018-23840-2

Figure Lengend Snippet: A proposed mechanism underlying the inactivation of STAT3 by curcumin. Covalent modification of STAT3 at its cysteine 259 residue by curcumin interrupts phosphorylation, dimerization and nuclear translocation of STAT3 and consequently induces apoptotic death in H- Ras MCF10A cells.

Article Snippet: Primary antibodies against P-STAT3 Y705 , STAT3, phosphorylated extracellular signal-regulated protein kinase (P-ERK) and phosphorylated JAK3 (P-JAK3) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).

Techniques: Modification, Residue, Phospho-proteomics, Translocation Assay

Journal: Psoriasis: Targets and Therapy

Article Title: TYK2 rs34536443 (P1104A) Variant Suppresses ICAM1-Mediated Inflammation: Insights From Mendelian Randomization and Functional Analyses

doi: 10.2147/PTT.S535434

Figure Lengend Snippet: Effect of TYK2-P1104A mutation and Deucravacitinib on STAT3 phosphorylation and ICAM1/ICAM5 expression. ( A ) Immunofluorescence analysis of the effect of TYK2-P1104A mutation on LPS-induced phosphorylation of STAT3 (p-STAT3) (magnification, 200×; scale bar, 50 µm) (n = 5). ( B ) Expression levels of ICAM1 and ICAM5 in lesional skin tissues from IMQ-induced psoriasis-like mouse models following Deucravacitinib treatment. Significant differences were denoted by ns (not significant), ** (P < 0.01), *** (P < 0.001) and **** (P < 0.0001).

Article Snippet: After antigen retrieval (eg, in sodium citrate buffer, pH 6.0, at 95–100 °C for 20 min) and blocking with immunohistochemical blocking solution (Beyotime, China) at 37 °C for 30 min, sections were incubated with primary antibodies against p-STAT3 (Y705) (GB150001-100, Servicebio, Wuhan, China), ICAM1 (GB11106-100, Servicebio, Wuhan, China), or ICAM5 (12,759-1-AP, Proteintech, Wuhan, China) at 37 °C for 45 min. After washing with PBS, sections were incubated with fluorophore-conjugated secondary antibodies (eg, Alexa Fluor 488- or 594-conjugated goat anti-rabbit IgG, Thermo Fisher Scientific, 1:500 dilution) at 37 °C for 45 min.

Techniques: Mutagenesis, Phospho-proteomics, Expressing, Immunofluorescence