BLT2-IL-8 Cascade in Aggressive Breast Cancer Figure 6. BLT2-dependent production of IL-8 contributes to the invasiveness of breast cancer cells. (A) MCF-10A, MDA-MB-231, and MDA-MB-435 cells were subjected to semiquantitative RT-PCR analysis of IL-8 mRNA (left panels). The cells (26105) per well in six-well plates were also incubated for 48 h, after which the amount of IL-8 released into the medium was measured with the use of a specific ELISA (right panel). (B) MDA-MB-231 and MDA-MB-435 cells were transfected with BLT2 or control siRNAs for 48 h, after which the secretion of IL-8 was determined as in (A). (C) MDA-MB-231 and MDA-MB-435 cells were incubated with Bay11-7082 (10 mM) or DMSO vehicle for 48 h, after which the amount of IL-8 released into the medium was determined. (D) 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 the secretion of IL-8 was determined as in (A). (E and F) MDA-MB-231 and MDA-MB-435 cells were transfected with sense (S) or antisense (AS) IL-8 oligonucleotides for 48 h, after which the amounts of IL-8 were determined by semiquantitative RT- PCR and ELISA assays (E) and the cells were assayed for invasiveness (F). Scale bars, 50 mm. Semiquantitative RT-PCR data are representative of three independent experiments and all quantitative data are means6SD from three independent experiments. *P,0.05, {P,0.01, {P,0.005. doi:10.1371/journal.pone.0049186.g006 These cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM glutamine, penicillin (100 U/ ml), and streptomycin (100 U/ml). Human immortalized mam- mary epithelial MCF-10A cells were kindly provided by A. Moon (Duksung Women’s University, Seoul, Korea) [33] and were maintained in DMEM/F12 supplemented with 5% heat-inacti- vated horse serum, insulin (10 mg/ml), epidermal growth factor (20 ng/ml), cholera enterotoxin (100 ng/ml), hydrocortisone (0.5 mg/ml), penicillin (100 U/ml), and streptomycin (100 U/ ml). All cells were maintained at 37uC under an atmosphere of 5% CO2. Semiquantitative RT-PCR Analysis of BLT1, BLT2, Nox1, Nox4, and IL-8 mRNAs Total RNA was extracted from cells with the use of Easy Blue (Intron, Sungnam, Korea), dissolved in diethylpyrocarbonate- treated water, and quantified by measurement of absorbance at 260 nm. The RNA (1.25 mg) was subjected to reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification of BLT2, Nox4, IL-8, and glyceraldehyde-3-phosphate dehydro- genase (GAPDH) cDNAs with the use of an RT-PCR PreMix Kit (Intron), as described previously [34]. PCR amplification of BLT1 cDNA was performed with the primers 59-TATGTCTGCG- PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49186