Figure 1

NPD1 upregulates Iduna expression in RPE cells undergoing UOS. (a) Experimental design timeline. Cells were transfected and recovered for 72 h. After 8 h of incubation in low serum medium (starvation), UOS was induced with 600 μM H₂O₂ plus 10 ng/ml of TNF-α in the presence and absence of NPD1. After a variable period (t=x) that ranged between 3 and 24 h (b) or 6 h (c–e and h), cells were harvested for western blot. For Hoechst staining, cells were fixed after 12 h of treatment (f and g). (b) Western blot of the time course of Iduna expression in ARPE-19 cells. (c and d) NPD1 (1–100 nM)-mediated expression of Iduna in UOS-treated ARPE-19 (c) and hRPE cells (d). (e) Specific expression of Iduna by NPD1, since the lipid mediators lipoxin A4 (LPX-A4) and 15-epi-LPX are inactive in ARPE-19 cells. (f) Representative images of Iduna expression by transient transfection of human Iduna construct (Iduna-146-GFP-Hu), (g) quantification of Hoechst-positive ARPE-19 cells and (h) western blot from transfection with Iduna human constructing ARPE-19 cells undergoing UOS and treated with 50 nM NPD1. Bars represent data averages of three repeats (technical replicas) of three independent experiments (biological replicas). *P<0.05