Dusp4 sirna

MCF-7 and MDA-MB-231 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM (Invitrogen, Life Technologies, Carlsbad, CA) supplemented with 10% FBS, 2mM L-glutamine, and 1% penicillin-streptomycin-neomycin. MCF-7 cells were stimulated with 1.32 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St Louis, MO) or 5 ng/ml recombinant TGF-β1 (R&D Systems, Minneapolis, MN) for 60 h, as previously described [28 ]. 0.65 nM triptolide (Santa Cruz, Dallas, TX) and 1 μM NSC 95397 (Santa Cruz) were used for inhibitor experiments. Forward transfection reactions were performed for 6 h with 50 nM human DUSP1 siRNA (sc-35937), DUSP4 siRNA (sc-38998), DUSP6 siRNA (sc-39000), 20 nM PKC-θ siRNA (sc-36252), and 10 nM MOCK siRNA (sc-36869) (Santa Cruz) using Lipofectamine 2000 (Invitrogen).

GC cells were transfected with DUSP4 siRNA (100 nmol/L; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or Twist siRNA (100 nmol/L; Santa Cruz) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Non-specific negative control siRNA was purchased from GeneChem Co., Ltd. (Shanghai, China). Six hours after the cells were transfected, the media was replaced with fresh culture media. All assays were performed 48 h after transfection and repeated three times.