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
Figure Lengend Snippet: TPA induces cellular β-catenin and STAT3α protein expression and nuclear translocation and β-catenin siRNA abolishes TPA-induced STAT3α and fascin-1 expression in MCF-7 cells 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
Article Snippet: Reagents 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, Translocation Assay, Western Blot, Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Positive Control