small interfering RNAs (siRNAs) were designed as described previously [12] and were obtained from Ambion (Austin, TX). RNAi for Nox1 was performed by transfection of cells with pSUPER vectors (SUPPLIER) as described previously [37]. Invasion Assays The invasion potential of MDA-MB-231 and MDA-MB-435 cells was assessed with the use of BioCoat Matrigel Invasion Chambers (BD Biosciences, Bedford, MA). Cells (56104) were harvested from culture plates by exposure to trypsin-EDTA, washed with RPMI 1640 supplemented with 0.5% FBS, and seeded in the same medium on the rehydrated Matrigel inserts. RPMI 1640 supplemented with 5% FBS was then added to the lower chamber as a chemoattractant, and the cells were incubated at 37uC for 36 h. Cells on the filters were subsequently fixed in methanol and stained with hematoxylin-eosin (H&E). Those on the upper surface of each filter were removed, and the invading cells were then counted in 10 random high-power (620) fields with the use of a CKX41 microscope (Olympus, Tokyo, Japan) equipped with a DP71 digital camera (Olympus). Each sample was assayed in triplicate, and the assays were performed five times. Measurement of ROS Measurement of intracellular H2O2 was performed as described previously [21]. The mean fluorescence intensity of dichlorofluor- escein was measured by flow cytometry with a FACSCalibur instrument (excitation, 488 nm; emission, 530 nm). Luciferase Reporter Assay for NF-kB Activity MDA-MB-231 and MDA-MB-435 cells were transfected with 1.4 mg NF-kB leuciferase reporter gene [38] using Lipofectamine reagent (Invitrogen, Carlsbad, CA), according to the manufactur- er’s instructions. To monitor variations in cell numbers and transfection efficiency, MDA-MB-231 and MDA-MB-435 cells were co-transfected with 0.6 mg pSV40-b-galactosidase, a eukary- otic expression vector containing the Escherichia coli b-galactosidase (lacZ) structural gene under the control of the SV40 promoter. After 24 h incubation in complete media, cells were incubated for 12 h in RPMI containing 0.5% FBS. The cells were then harvested and assayed for luciferase activity as described [38]. Transfection with an Antisense Oligonucleotide for IL-8 Oligonucleotides were obtained from Bioneer (Daejeon, Korea). An antisense oligonucleotide for IL-8 was designed as previously described [39]. Cells were plated at a density of 46105 per 60-mm dish, cultured for 24 h, and transfected for 36 h with 100 nM of antisense or control sense oligonucleotides with the use of Lipofectamine (Invitrogen). The depletion of IL-8 mRNA in the cells was assessed by RT-PCR analysis. Immunoblot Analysis MDA-MB-231 and MDA-MB-435 cells were collected and lysed in lysis buffer [40 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.1% Nonidet-P40, 100 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2 mg/ml leupeptin, 2 mg/ml aprotinin]. Proteins in the lysate were separated by SDS-PAGE and transferred onto a nitrocellulose membrane, after which the membrane was blocked with 5% nonfat dry milk in Tris-buffered saline and then incubated with primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against p-IkB-a and a-tubulin for 1 h at room temperature. Blots were developed with a peroxidase- conjugated secondary antibody, and proteins were visualized using BLT2-IL-8 Cascade in Aggressive Breast Cancer ECL reagents (Amersham, Arlington Heights, IL) according to the manufacturer’s recommendations. ELISA for IL-8 Measurement An enzyme-linked immunosorbent assay (ELISA) kit for human IL-8 was obtained from BD Biosciences. In vivo Metastasis Assays The study was approved by the ethics committee of Korea University, and all experimental animals used in this study were treated according to the guidelines approved by the Institutional Animal Care and Use Committee of Korea University. Female nude mice (Charles River, Wilmington, MA) were used for metastasis assays and were 6 weeks old at the time of cell injection. For experimental metastasis assays, cultured MDA-MB-231 cells (56105) were pretreated with LY255283 or DMSO for 24 h to ensure the inhibition of BLT2 signaling and were then suspended in PBS and injected into the tail vein. LY255283 (2.5 mg/kg) or DMSO vehicle was injected intraperitoneally 3 and 5 days after cell injection. The mice (n = 4 for each group) were maintained under aseptic barrier conditions until they were killed at 10 weeks after cell injection for detection of pulmonary metastases. The number of lung surface metastasis nodules with a diameter of .0.2 mm was determined. For spontaneous metastasis assays, mice (n = 3 for each group) were anesthetized by intraperitoneal injection of zoletil (50 mg/kg) and xylazine (2.5 mg/kg), and then MDA-MB-231 cells (26106) pretreated with LY255283 or DMSO for 24 h in PBS were implanted into the mammary fat pad. LY255283 (2.5 mg/kg) or DMSO vehicle was injected intraper- itoneally three times at 5-day intervals beginning immediately after cell implantation. The mice were killed and subjected to necropsy at 14 weeks after cell implantation. The primary tumors were removed, and the presence of lung metastases was determined by microscopic analysis. The lung and tumor tissue were fixed in 4% formalin, embedded in paraffin, sectioned, and stained with H&E for examination with a BX51 microscope (Olympus) equipped with a DP71 digital camera (Olympus). Data Analysis and Statistics The results are presented as mean6SD. Analyses were performed with the Student t test using SigmaPlot 8.0. Values of p,0.05 were considered to be significant. Supporting Information Figure S1 Blockade of BLT2 with LY255283 or RNAi does not affect the proliferation or survival. (A and B) Cells transfected with control or BLT2 siRNAs for 24 h were incubated either for the indicated times for determination of cell growth by trypan blue staining (A) or for 48 h for determination of cell survival by flow cytometric analysis of the FITC-Annexin V/ PI staining and the sub-G1 population (B). (C and D) Cells exposed to U75302 (0.5 or 1 mM), LY255283 (5 or 10 mM), or DMSO vehicle for the indicated times (C) or for 48 h (D) were assayed for cell growth and apoptosis, respectively. All data are means6SD from three independent experiments. (TIF) Figure S2 Knockdown of Nox1 by RNAi. MDA-MB-231 and MDA-MB-435 cells were transfected with a vector for Nox1 siRNA (pSUPER-siNox1) or the corresponding empty vector for 48 h, after which total RNA was isolated from the cells and the amount of Nox1 mRNA was determined by semiquantitative RT- PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49186