FIGURE 3.

Cyclic stretch does not alter the LPS-induced NF-κB signaling pathway. (A): J774.1 cells were exposed to CS of 20% elongation at a frequency of 10 cycles/min with 100 ng/ml LPS for the indicated times. Cell lysates were analyzed by Western blotting with anti-IκB-α. An antibody against β-actin was used as a control. Results are representative of three independent experiments. (B–D): J774.1 cells were exposed to CS of 20% elongation at a frequency of 10 cycles/min for the first 2 h during treatment with 100 ng/ml LPS for 4 h. (B): The nuclear translocation of NF-κB p65 was detected by immunostaining (green: shown in the upper panel). Nuclei were visualized by staining with DAPI (blue: shown in the middle panel). Merged images are shown in the lower panel (magnification: × 400; scale bars are 20 μm). White arrows indicate p65 nuclear translocation. Results are representative of three independent experiments. (C): Cells showing the nuclear translocation of NF-κB p65 in three randomly selected fields (each containing ∼100 cells) were quantified. Representative data of three separate experiments are shown as the means ± SD of triplicate assays (ns; not significant). (D): Nuclear proteins were extracted from cells and a NF-κB ELISA assay was performed. The positive control (Pos.) was provided by a nuclear extract (5 μg) of Raji cells. A sample with no cell extract was used as a negative control (Neg.). Representative data of three separate experiments are shown as the means ± SD of triplicate assays (ns; not significant).