Activation of the leukotriene B4 receptor 2-reactive oxygen species (BLT2-ROS) cascade following detachment confers anoikis resistance in prostate cancer cells - PubMed (original) (raw)

NOX-dependent ROS generation is downstream of BLT2 signaling. A, PC-3 cells were incubated on poly-HEMA-coated plates for the indicated times and then subjected to semiquantitative RT-PCR. B, PC-3 cells were detached and maintained for the indicated times in suspension or adherent culture. NOX1 and NOX4 mRNA expression levels were assessed by real-time PCR. *, p < 0.05; **, p < 0.01; ***, p < 0.005. C–E, PC-3 cells were transfected with control (scrambled) or 20 n

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BLT2 siRNA. After 36 h, the cells were detached and maintained for an additional 3 h in suspension or adherent culture. NOX1 and NOX4 mRNA expression levels were determined by semiquantitative RT-PCR (C) and quantitative real-time PCR (D). The intracellular ROS levels were determined by measuring DCFDA fluorescence with a flow cytometer (E). *, p < 0.05; ***, p < 0.005. F, PC-3 cells were detached, treated with DMSO or baicalein (10 μ

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), and maintained for 3 h in suspension or adherent culture. The ROS levels were analyzed by flow cytometry. ***, p < 0.005. G, PC-3 cells were treated with DMSO or DPI (100 n

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) for 24 h in suspension or adherent culture, and cell viability was determined using a WST-1 assay. *, p < 0.05. H, PC-3 cells were treated with DMSO or DPI (100 n

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). After 1 h, the cells were seeded onto soft agar plates. After 3 weeks, the colonies were stained with crystal violet and examined under a microscope. *, p < 0.05. I, PC-3 cells were transfected with 2 μg of pSuper (control), pSuper-siNOX1, or pSuper-siNOX4 for 24 h using Lipofectamine 2000. Then, the cells were detached and maintained for an additional 3 h in suspension or adherent culture. NOX1 and NOX4 mRNA expression levels were determined by semiquantitative RT-PCR (left panel), and the intracellular ROS levels were measured by flow cytometry (right panel). *, p < 0.05; ***, p < 0.005. J, PC-3 cells were transfected with pSuper (control), pSuper-siNOX1, or pSuper-siNOX4 and detached as in I. After 24 h, the cells were subjected to a WST-1 assay. *, p < 0.05; ***, p < 0.005. K, PWR-1E cells were transfected with pcDNA3.1, pcDNA3.1-NOX1, or pcDNA3.1-NOX4 for 36 h. Then, the cells were maintained for an additional 24 h in suspension or adherent culture, and the cell viability was determined using a WST-1 assay. *, p < 0.05. All of the quantitative data are shown as the mean ± S.D. (error bars) of three independent experiments.