Figure 3

NPD1 mediates expression and translocation of Iduna and PAR dependency. (a) Effect of NPD1 on apoptosis in ARPE-19 cells transfected with either shRNA-Iduna or YRAA-Iduna construct. (b) Western blot and (c) densitometric quantification of Iduna expression by NPD1 in ARPE-19 cells and controls transfected with shRNA-Iduna and YRAA-Iduna construct. (d and e) Silencing of Iduna using two different siRNAs (siRNA1 and siRNA2) and a control nonspecific targeting siRNA (scramble). (d) Hoechst staining of transfected or non-transfected ARPE-19 cells undergoing UOS in the presence or absence of NPD1 and (e) Western blot and densitometry of Iduna expression. (f) Endogenous expression of Iduna in response to NPD1. Representative images of immunostaining of ARPE-19 cells undergoing UOS in the absence or presence of 50 or 100 nM NPD1 and quantification of Iduna-positive cells. (f and h) Arrows mark examples of the Iduna-positive nuclei. (g and h) Nuclear translocation of Iduna by NPD1. (g) Western blot analysis and densitometry of whole cell (WC), cytoplasmic (Cyto) and nuclear fractions of ARPE-19 cells undergoing UOS in the presence or absence of 100 nM NPD1. Whole cell and cytoplasmic fractions where standardized using GAPDH and nuclear fraction using TBP. (h) Z-stacks flattened 2D images (red=DAPI first column; green=Iduna, second column; merged channels, third column) and 3D representation of ARPE-19 cells immunocytochemistry targeting endogenous Iduna. Bar=200 μm. Arrows indicate co-localization (yellow) of both the DAPI and Iduna signal in the nucleus. Bars represent data averages of three repeats (technical replicas) of three independent experiments (biological replicas). *P<0.05