Figure 8. Scheme for the involvement of BLT2 cascade in IL-8 production and invasiveness of breast cancer cell. BLT2-induced IL-8 up-regulation leads to enhanced invasiveness of breast cancer cells. The activation of the BLT2 pathway induced Nox1-dependent ROS generation. Then, these ROS may contribute to NF-kB activation, which lead to IL-8-dependent invasiveness. doi:10.1371/journal.pone.0049186.g008 GAGTCAGCATGTACGC-39 (sense) and 59-CCTGTAGCC- GACGCCCTATGTCCG-39 (antisense). Amplification was con- firmed to be in the linear range of the reaction. The amplification protocol for BLT1 cDNA included 30 cycles of denaturation at 95uC for 30 s, annealing at 67uC for 20 s, and elongation at 72uC for 40 s. Analysis of Nox1 mRNA was performed by RT and a two-step PCR procedure as described previously [35]. RT was thus performed with the reverse primer of the first-round PCR (see below), and cDNA derived from 1.25 mg of total RNA was subjected to the first-round PCR with the forward Nox1 primer 59-CAGGGAGACAGGTGCCTTTTCC-39 (beginning at nucle- otide 1523, GenBank accession no. NM 007052) and the reverse Nox1 primer 59-GCTCAAACCTGACGAGACCAAG-39 (ending at nucleotide 2037). For the second-round, nested PCR, the forward nested primer was 59-AACCTGTTGACTTCCCTG- GAAC-39 (beginning at nucleotide 1554) and the reverse nested primer was 59-TCCAGACTGGAATATCGGTGAC-39 (ending at nucleotide 1858). The amplification protocol for both first- round and nested PCR included 27 cycles of denaturation at 95uC for 30 s, annealing at 61uC for 40 s, and elongation at 72uC for 45 s. The specificity of all primers was confirmed by sequence analysis of the PCR products. RT and Real-time Quantitative PCR Analysis of BLT1 and BLT2 mRNAs Total RNA (1.25 mg) extracted from cells with the use of Easy Blue (Intron) was subjected to RT with Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA), and BLT1, BLT2, and GAPDH cDNAs were amplified as described previously [13] with the use of a LightCycler 480 SYBR Green I Master instrument (Roche, Mannhein, Germany). Melting curves were analyzed to ensure amplification specificity for the PCR products. The amounts of BLT1 and BLT2 mRNAs were normalized by the corresponding amount of GAPDH mRNA. BLT2-IL-8 Cascade in Aggressive Breast Cancer Flow Cytometric Analysis of BLT2 For quantitation of BLT2 expression on the cell surface, cells were incubated in 60-mm dishes for 24 h. The cells were detached by treatment with trypsin, washed with PBS and fixed in 2% paraformaldehyde. After exposure to 2% bovine serum albumin for 30 min, the cells were incubated for 1 h at room temperature with rabbit polyclonal antibodies to BLT2 (MBL-2097, 1:100 dilution; Life Span, Des Plaines, IL), washed three times with phosphate-buffered saline (PBS), and incubated for 30 min at room temperature with fluorescein isothiocyanate–conjugated goat antibodies to rabbit immunoglobulin G (1:200 dilution; Molecular Probes, Eugene, OR). The cells (10,000 per sample) were then subjected to flow cytometry with the use of a FACSCalibur instrument and Cell Quest software (Becton Dickinson, Franklin Lakes, NJ) for determination of mean fluorescence intensity. Cell Growth Assay Cells were plated at a density of 0.56105 or 26105 cells per well in 12-well plates and cultured for 24 h. Cells were transfected with control or BLT2 siRNAs for 24 h, after which they were incubated in medium supplemented with 0.5% FBS and incubated for the indicated times. Also, cultured cells were incubated in medium supplemented with 0.5% FBS for 3 h and then exposed to U75302 (0.5 or 1 mM) or LY255283 (5 or 10 mM) and incubated at 37uC for the indicated times. The cells were then isolated by exposure to trypsin-EDTA, and the number of viable cells was determined by staining with trypan blue and light microscopy. Detection of Apoptosis via FITC-Annexin V/PI Saining Cells were plated at a density of 36105 cells per well in 6-well plates and cultured for 24 h. The cells were transfected with control or BLT2 siRNAs for 24 h, after which they were incubated in medium supplemented with 0.5% FBS and incubated for 48 h. Cells (16105) were collected and suspended in 100 ml 16Annexin V Binding Buffer (BD Bioscience, San Jose, CA). 2 ml FITC- Annexin V (BD Bioscience) were added as well as 10 ml propidium iodide (PI) (Sigma, Saint Louis, MO) staining to a final concentration of 5 mg/ml and the cells were incubated at room temperature for 15 min in the dark. Then, 400 ml of Annexin V binding buffer were added and flow cytometry was performed using a BD FACSCalibur flow cytometer. Cells were considered to be apoptotic if they were Annexin V+/PI– (early apoptotic) and Annexin V+/PI+ (late apoptotic). Each analysis was performed using at least 20,000 events. Four independent experiments were performed. Flow Cytometric Analysis of Sub-G1 Population The sub-G1 (apoptotic) population was assessed as described [36]. Cells treated as described for the cell growth assay were fixed in 70% ethanol, resuspended in 0.5 ml of PBS, mixed with 0.5 ml of DNA extraction buffer (19.2 mM Na2HPO4, 0.004% Triton X- 100), and incubated for 5 min at room temperature. The cells were then isolated by centrifugation at 4006g for 5 min, stained for 30 min with PI (50 mg/ml) in PBS containing RNase A (100 mg/ml), and analyzed by flow cytometry with a FACScan instrument and CellQuest software (Becton Dickinson). Cells with a DNA content less than that of cells in G1 phase (sub-G1) were assumed to be apoptotic. RNA Interference (RNAi) Depletion of BLT2 mRNA by RNAi was performed as described previously [13]. BLT2-specific and scrambled control PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49186