FIGURE 13.

Analyses of NF-κB properties in ECs stimulated by 15d-PGJ₂. A, HUVECs were treated with 10 μm 15d-PGJ₂ for the indicated time periods. Cells were harvested and subjected to Western blot analysis using phospho-specific antisera to NF-κB or IκBα and then probing membranes with total NF-κB or IκBα antibody. Asterisk indicates cells were treated with 10 mm NAC for 30 min prior to treatment with 15d-PGJ₂. Shown are blots representative of at least three independent experiments. B, NF-κB activity. Nuclear extracts from 5 × 10⁵ HUVECs were measured for the NF-κB DNA binding activity by ELISA (n = 4). ^*, p < 0.05 versus untreated (UT) cells. #, p < 0.005 versus 15d-PGJ₂-treated cells. C, HUVECs were treated with inhibitors of NF-κB(5 μm; 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) or 10 μm SN50, 1 h) before exposure to 15d-PGJ₂ for a further 16 h, and then apoptosis was examined by counting the annexin V-positive cells. ^*, p < 0.05 versus 15d-PGJ₂-treated cells. D, 15d-PGJ₂ effect on the cellular distribution of NF-κB p65 subunit in vascular ECs. Mouse corneas were subjected to alkali trauma and eyes were treated 3 days later with either 20 μl of PBS or 20 μm 15d-PGJ₂. After treatment for 8 h, corneas were harvested and subjected to immunofluorescence for eNOS (red) and p65 (green). Nuclei were stained by Hoechst 33342 (blue). Magnification, ×40. Bar, 75 μm.