Figure 1
IL-8 protein and mRNA expression in cell lines A2058, A2058T2-1, and A2058T2R-7. (A) IL-8 secretion in cell lines A2058, A2058T2-1, and A2058T2R-7. Equal number of cells was plated in 6-well plates. After growing for 24 hours, the supernatant was collected and assayed for IL-8 ELISA. Data are the mean ± SD of a single experiment assayed in triplicate and is representative of 3 separate experiments. **P < .001 for A2058 vs A2058T2-1. (B) IL-8 mRNA expressionin the A2058, A2058T2-1, and A2058T2R-7 cells. Total RNA was extracted using TRIzol reagent and the RNA integrity was verified by electrophoresis gel. All IL-8 expression levels were normalized to the GAPDH levels of the same sample. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control cells. All real-time PCR reactions were performed in triplicate. **P < .001 for A2058 vs A2058T2-1.
Figure 2
Immunoblot of whole cell IκBα and phospho-IκBα in cell lines A2058, A2058T2-1, and A2058T2R-7. (A) Western blot of TIMP-2, IκBα, and phospho-IκBα. (B) Densitometry of IκBα and phospho-IκBα in cell lines A2058, A2058T2-1, and A2058T2R-7. Relative band intensity was determined using NIH Image 1.63 software. Data are reported as mean ± SD of 3 independent experiments.
Figure 3
Immunoblot of whole cell NF-κBp65 and phospho- p65 in cell lines A2058, A2058T2-1, and A2058T2R-7. (A) Western blot of p65 and phospho-p65; (B) Densitometry of p65 and phospho-p65. Relative band intensity was determined using NIH Image 1.63 software. Data are reported as mean ± SD of 3 independent experiments.
Figure 4
Transcriptional activity of NF-κB in cell lines A2058, A2058T2-1, and A2058T2R-7. NF-κB transcription was measured by luciferase activity. Data are reported as mean ± SD of a single experiment assayed in triplicate and is representative of 3 separate experiments. ** p < 0.001 for A2058 group vs A2058T2-1 or A2058T2R-7 vs A2058T2-1.
Figure 5
Apoptosis in A2058, A2058T2-1, and A2058T2R-7 with or without TNF-α treatment. After 6 h of treatment with TNF, 1 × 106 adherent cells were trypsinized and incubated with FITC-conjugated annexin V and propidium iodide (PI) for 15 min in the dark. Cells were analyzed by flow cytometry. The percentages of cells in each groups within the gated areas are indicated. In the gate areas, the up-right panel is for the population of later apoptotic cells and the low-right panel is for the early apoptosis cells. Dot plots of early apoptotic cells have increased Annexin VFITC fluorescence only, whereas necrotic and late apoptotic cells have increased annexin V and PI fluorescence. Axes are labelled with arbitrary fluorescence units. Data are from a representative of three independent experiments that gave similar results.