The cells were incubated using the transfection mixture for 6 h and then rinsed with medium containing 10% FBS

The cells were incubated using the transfection mixture for 6 h and then rinsed with medium containing 10% FBS. We further found that both CUG2-induced TGF- production and TGF–induced CUG2 up-regulation required a physical interaction between Sp1 and Smad2/3 in the CUG2 and TGF- promoter, as demonstrated by a promoter reporter assay, immunoprecipitation, and ChIP assay. These results indicated close crosstalk between CUG2 and TGF-. Conversely, suppression of CUG2 or NPM1 did not completely inhibit TGF–induced EMT, indicating that the effect of TGF- on EMT is dominant over the effect of CUG2 on EMT. Collectively, our findings suggest that CUG2 induces the EMT via TGF- signaling. 0.001, except **; for 15 min. The supernatant was taken as the soluble fraction, and the pellets as insoluble fractions were subsequently solubilized in 800 L of RIPA buffer (50 mM Tris-HCl [pH7.4], 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 10% glycerol, 0.5 mM PMSF, and 1x protease inhibitor cocktail) on ice for 30 min and were centrifuged at 12,000for 15 min. Thereafter, the supernatants were used for the nuclear extracts. Immunoblotting and Immunoprecipitation Cells were harvested and lysed with lysis buffer containing 1% NP-40 and protease inhibitors (Sigma-Aldrich). For immunoblotting, proteins from whole cell lysates were resolved by 10% or 15% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes. Primary antibodies were used at a 1:1000 or 1:2000 dilution, and secondary antibodies conjugated with horseradish peroxidase were used at a 1:2000 dilution in 5% nonfat dry milk. For immunoprecipitation, cells were harvested after 48 h of transfection, and the cell debris was removed by centrifugation at 10,000 for 10 min at 4C. Cell lysates were pre-cleared with 25 L of protein A/G agarose and incubated with the appropriate primary antibody and protein A/G agarose for 1 h at 4C. After 3 washes with lysis buffer, the precipitates were resolved on SDS-PAGE gels and analyzed by immunoblotting with the appropriate antibodies. After the final washing, the membranes were evaluated with an enhanced chemiluminescence assay using the Image Quant LAS 4000 Mini (GE-Healthcare, Tokyo, Japan). Luciferase reporter assays A549-Vec, A549-CUG2, BEAS-Vec, and BEAS-CUG2 cells were transfected with TGF- promoter vectors (phTG1, 5, 6, 7, and 7-4) [41], or CUG2 promoter vectors (F961 and F961-94)[23] with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). To normalize transfection efficiency, a pGK-gal vector that expresses -galactosidase under a control of a phosphoglucokinase promoter was included in the transfection mixture. At 48 h post-transfection, cells were washed with cold PBS and lysed in lysis solution (25 mM Tris [pH7.8], 2 mM EDTA, 2 mM DTT, 10% glycerol, and 1% Triton-X100). Luciferase activity was measured with a luminometer by using a luciferase kit (Promega, Madison, WI). Short interfering RNA (siRNA) transfection Cells were trypsinized and cultured overnight to achieve 60-70% confluency before siRNA transfection. NPM1 siRNAs (#1 ; AAC ACC ACC AGU GGU CUU AAG, # HC-030031 2# 2 ; GAA AAU GAG CAC CAG UUA U, Bioneer, Daejeon, Korea), pre-made CUG2 siRNA (Bioneer), TGF-1 siRNA (Bioneer), or a negative control siRNA (Bioneer) were mixed with Lipofectamine 2000. The cells were incubated with the transfection mixture for 6 h and then rinsed with medium containing 10% FBS. The cells were incubated for 48 h before harvesting. Invasion assay Invasion assays were performed using 48-well Boyden chambers (Neuroprobe, Gaithersburg, MD) as described elsewhere [42]. The lower wells of the chamber were filled with standard culture media. The chamber was assembled using polycarbonate filters (Neuroprobe) coated with Matrigel. Cells in serum-free HC-030031 media (5104 cells per well) were seeded in the upper compartment of the chamber. After incubation for 24 h, cell migration was quantified by counting the number of migrated cells after staining with hematoxylin-eosin. Wound healing assay Cell migration was assessed using a scratch wound assay [43]. Briefly, the cells were cultured in six-well plates PRPF10 (5 105 cells per well). When the cells were reached 90% confluence, a single wound was made in the center of HC-030031 the cell monolayer using a P-200 pipette tip. At 0 and 24 h of incubation, the wound closure areas were visualized by phase-contrast microscopy (Olympus, CKX31-11 PHP, Tokyo,.