Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: After treatment with various concentrations of TPA for 24 h ( A ) or with 100 ng/ml of TPA for 0–24 h ( B ), cellular β-catenin, STAT3α, and fascin-1 expression as well as nuclear (N) β-catenin and STAT3α expression were determined by Western blotting. ( C ) Cells were transfected with β-catenin siRNA or nontargeting control (NTC) and were then treated with 100 ng/ml of TPA for an additional 24 h. β-catenin, STAT3α, and fascin-1 proteins were determined. ( D ) MCF-7 cells were treated with 100 ng/ml of TPA for 6 h, and cell lysate was prepared for ChIP-PCR assay for STAT3 binding in fascin-1 gene in MCF-7 cells. “Input”, total input DNA; “H3”, DNA-protein complex pulled down by acetyl Histone H3; “STAT3”, DNA-protein complex pulled down by anti-STAT3; and “IgG”, DNA-protein complex pulled down by rabbit IgG antibody. H3 served as a positive control for STAT3 binding. Values are mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Expressing, Western Blot, Transfection, Control, Binding Assay, Positive Control

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: MCF-7 cells were treated with various concentrations of TPA for 30 min ( A ), were treated with 100 ng/ml of TPA for different time periods ( B ), or were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 30 min ( C ). STAT3α phosphorylation at Tyr705 was measured. Cells were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 24 h. The amounts of cellular β-catenin protein ( D ) were determined. Changes in β-catenin ( E ) and fascin-1 mRNA ( F ) and protein ( G ) were measured in cells pretreated with or without 5 μM WP1066 for 4 h, followed by incubation with TPA for another 18 h and 24 h, respectively. ( H ) Cells were transfected with STAT3 expression vector or pcDNA3.1 ( − ) control vector and were then treated with 100 ng/ml of TPA for an additional 24 h. STAT3α, β-catenin, and fascin-1 proteins were determined. One representative experiment out of three independent experiments is shown. Values are presented as mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Incubation, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: MCF-7 cells were treated with various concentrations of TPA for 30 min and PKCδ in the plasma membrane and cytosolic fractions were determined ( A ). Cells were pretreated with or without 0.5 μM nonselective PKC inhibitor GF109203X (GF) or 5 μM PKCδ-specific inhibitor rottlerin (Rot) for 1 h followed by incubation with 100 ng/ml of TPA for another 30 min. STAT3α and GSK3β phosphorylation were measured ( B ). β-Catenin, STAT3α, and fascin-1 mRNA ( C ) and protein ( D ) levels were determined after 18 h and 24 h with TPA treatment, respectively. One representative experiment out of three independent experiments is shown. Mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Clinical Proteomics, Membrane, Incubation, Phospho-proteomics

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: MCF-7 cells were pretreated with 0, 25, or 100 μM DHA for 24 h followed by incubation with 100 ng/ml of TPA for another 30 min. PKCδ in the plasma membrane and cytosol ( A ) and STAT3α ( B ) and GSK3β ( C ) phosphorylation were determined. ( D ) Total cellular β-catenin and STAT3α and nuclear β-catenin protein levels were determined in cells pretreated with various concentrations of DHA for 24 h followed by incubation with TPA for another 24 h and 4 h, respectively. ( E ) Cells were pretreated with 100 μM DHA for 24 h, 5 μM WP for 4 h, or 5 μM rottlerin (Rot) for 1 h, and then were incubated with TPA for another 6 h. Nuclear extracts (10 μg) were prepared for STAT3 nuclear protein DNA binding activity assay. To confirm the specificity of the nucleotide, 50-fold cold probe and mutant (Mu) were included in the EMSA. One representative experiment out of three independent experiments is shown. ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Incubation, Clinical Proteomics, Membrane, Phospho-proteomics, Binding Assay, Activity Assay, Mutagenesis

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: ( A ) β-catenin, STAT3α, and fascin-1 expression in MCF-7 and Hs578T cells were determined by Western blotting. ( B ) Hs578T cells were treated with various concentrations of DHA for 24 h. ( C ) Cells were transiently transfected with β-catenin, STAT3, and fascin-1 siRNA or with nontargeting control (NTC) followed by treated with or without 100 μM DHA, 5 μM Rot or 5 μM WP1066 for an additional 24 h. Protein levels of β-catenin, STAT3α, and fascin-1 (C) as well as cell migration ( D ) were determined. One representative experiment out of three independent experiments is shown.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Expressing, Western Blot, Transfection, Control, Migration

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: After treatment with various concentrations of TPA for 24 h ( A ) or with 100 ng/ml of TPA for 0–24 h ( B ), cellular β-catenin, STAT3α, and fascin-1 expression as well as nuclear (N) β-catenin and STAT3α expression were determined by Western blotting. ( C ) Cells were transfected with β-catenin siRNA or nontargeting control (NTC) and were then treated with 100 ng/ml of TPA for an additional 24 h. β-catenin, STAT3α, and fascin-1 proteins were determined. ( D ) MCF-7 cells were treated with 100 ng/ml of TPA for 6 h, and cell lysate was prepared for ChIP-PCR assay for STAT3 binding in fascin-1 gene in MCF-7 cells. “Input”, total input DNA; “H3”, DNA-protein complex pulled down by acetyl Histone H3; “STAT3”, DNA-protein complex pulled down by anti-STAT3; and “IgG”, DNA-protein complex pulled down by rabbit IgG antibody. H3 served as a positive control for STAT3 binding. Values are mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Expressing, Western Blot, Transfection, Control, Binding Assay, Positive Control

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: MCF-7 cells were treated with various concentrations of TPA for 30 min ( A ), were treated with 100 ng/ml of TPA for different time periods ( B ), or were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 30 min ( C ). STAT3α phosphorylation at Tyr705 was measured. Cells were pretreated with 1 or 5 μM WP1066 for 4 h followed by incubation with 100 ng/ml of TPA for another 24 h. The amounts of cellular β-catenin protein ( D ) were determined. Changes in β-catenin ( E ) and fascin-1 mRNA ( F ) and protein ( G ) were measured in cells pretreated with or without 5 μM WP1066 for 4 h, followed by incubation with TPA for another 18 h and 24 h, respectively. ( H ) Cells were transfected with STAT3 expression vector or pcDNA3.1 ( − ) control vector and were then treated with 100 ng/ml of TPA for an additional 24 h. STAT3α, β-catenin, and fascin-1 proteins were determined. One representative experiment out of three independent experiments is shown. Values are presented as mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Incubation, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: MCF-7 cells were treated with various concentrations of TPA for 30 min and PKCδ in the plasma membrane and cytosolic fractions were determined ( A ). Cells were pretreated with or without 0.5 μM nonselective PKC inhibitor GF109203X (GF) or 5 μM PKCδ-specific inhibitor rottlerin (Rot) for 1 h followed by incubation with 100 ng/ml of TPA for another 30 min. STAT3α and GSK3β phosphorylation were measured ( B ). β-Catenin, STAT3α, and fascin-1 mRNA ( C ) and protein ( D ) levels were determined after 18 h and 24 h with TPA treatment, respectively. One representative experiment out of three independent experiments is shown. Mean ± SD, n = 3. * p < 0.05 and ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Clinical Proteomics, Membrane, Incubation, Phospho-proteomics

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: MCF-7 cells were pretreated with 0, 25, or 100 μM DHA for 24 h followed by incubation with 100 ng/ml of TPA for another 30 min. PKCδ in the plasma membrane and cytosol ( A ) and STAT3α ( B ) and GSK3β ( C ) phosphorylation were determined. ( D ) Total cellular β-catenin and STAT3α and nuclear β-catenin protein levels were determined in cells pretreated with various concentrations of DHA for 24 h followed by incubation with TPA for another 24 h and 4 h, respectively. ( E ) Cells were pretreated with 100 μM DHA for 24 h, 5 μM WP for 4 h, or 5 μM rottlerin (Rot) for 1 h, and then were incubated with TPA for another 6 h. Nuclear extracts (10 μg) were prepared for STAT3 nuclear protein DNA binding activity assay. To confirm the specificity of the nucleotide, 50-fold cold probe and mutant (Mu) were included in the EMSA. One representative experiment out of three independent experiments is shown. ** p < 0.01.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Incubation, Clinical Proteomics, Membrane, Phospho-proteomics, Binding Assay, Activity Assay, Mutagenesis

Journal: Oncotarget

Article Title: Docosahexaenoic acid inhibits 12- O -tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways

doi: 10.18632/oncotarget.7301

Figure Lengend Snippet: ( A ) β-catenin, STAT3α, and fascin-1 expression in MCF-7 and Hs578T cells were determined by Western blotting. ( B ) Hs578T cells were treated with various concentrations of DHA for 24 h. ( C ) Cells were transiently transfected with β-catenin, STAT3, and fascin-1 siRNA or with nontargeting control (NTC) followed by treated with or without 100 μM DHA, 5 μM Rot or 5 μM WP1066 for an additional 24 h. Protein levels of β-catenin, STAT3α, and fascin-1 (C) as well as cell migration ( D ) were determined. One representative experiment out of three independent experiments is shown.

Article Snippet: DMEM, fetal bovine serum (FBS), penicillin–streptomycin solution, and 25% trypsin–EDTA were from GIBCO-BRL (GIBCO, Gaithersburg, MD); albumin, essentially fatty acid–free bovine serum albumin (BSA), sodium bicarbonate, calcium chloride, MTT, GF109203X, rottlerin and TPA were from Sigma-Aldrich (St. Louis, MO); STAT3 inhibitor III (WP1066) was from Merck (Darmstadt, Germany); DHA was from Cayman Chemical (Ann Arbor, MI); TRIzol reagent, Opti-MEM medium, and Lipofectamine RNAi MAX transfection reagent were from Invitrogen (Carlsbad, CA); antibodies against PKCδ (GTX61806; 78 KDa), Wnt-1 (GTX111182; 41 KDa), fascin-1 (GTX10051; 55 KDa), and β-actin (GTX109639; 42 KDa) were from GeneTex (Irvine, CA); antibodies against phospho-STAT3α (Tyr705)(#9138; 86 KDa), STAT3α (#9139; 86 KDa) and PARP (#9532; 116 KDa) were from Cell Signaling Technology (Danvers, MA); antibodies against β-catenin (#06–734; 92 KDa), GSK3β (#05–903; 47 KDa), phospho-GSK3β (Ser9)(#05–643; 47 KDa), STAT3 (#06–596), and EZ-Magna ChIP assay kit (#17–408) were from Millipore (Billerica, MA); antibody against clathrin (sc-6579; 192 KDa) was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) and the KAPA SYBR FAST qPCR Kit was from KapaBiosystems (Woburn, MA).

Techniques: Expressing, Western Blot, Transfection, Control, Migration