Fig. 5.

NPD1 synthesis is mediated by 15-lipoxygenase-1. A: Immunocytochemistry showing localization of 15-LOX-1 in ARPE-19 cells. Right column shows normal cells and left column shows 15-LOX-1 silenced cells. The four upper panels depict the localization of 15-LOX1 (green) relative to the nuclei (blue) and Actin (red). The four lower panels display nuclear localization of 15-LOX-2 (red) in relationship with nuclei (blue) and Actin (green). B: Histograms showing the differential production of NPD1 upon different strength of oxidative stress treatment (0, 400, 600, 800 μM H₂O₂ and 10 ng/ml TNFα). In each cell, as in the medium, the production of NPD1 was almost completely abolished (^*P < 0.01). C: Apoptosis percentage measured by Hoechst staining of ARPE-19 cultures of control and silenced cells subjected to oxidative stress and treated with different metabolites of 15-LOX-1 and NPD1 precursor DHA. In silenced cells, apoptosis was augmented by oxidative stress. In normal cells, PEDF/DHA, NPD1, and lipoxin A4 did prevent apoptotic cell death, but in the silenced cells, neither DHA nor lipoxin A4 had any effect. Only NPD1 was able to rescue these cells from apoptosis. Data represents average ± SEM of two independent studies; statistical analysis is Student's t-test (^*P < 0.01, ^**P < 0.001, ^***P < 0.0001 and ^****P < 0.00001). Modified with permission from reference (91).