FIG. 4.

Role of Prx1 in the OGD-induced apoptotic cascade in endothelial cells. (A) The effects of Prx1 on OGD-induced ER stress signaling were determined by immunoblotting. (B) Densitometry of Western blots for PERK, Ire-1α, and calnexin levels 6 h after OGD, with or without Prx1 transfection. Data are expressed as densitometry ratio of control (mean±SEM). *p<0.05; ***p<0.001 versus control; ^#p<0.05 versus OGD. (C) The effects of Prx1 overexpression on OGD-induced caspase-3 and PARP levels were evaluated by immunoblotting. EA.hy926 cells were cultured and transfected with plasmid DNA encoding Prx1 or an empty plasmid using Attractene. (D) The quantification data for blots are shown in (C). Data are expressed as densitometry ratio of control (mean±SEM). **p<0.01 versus control; ^#p<0.05; ^##p<0.01 versus OGD. (E) The effects of Prx1 overexpression on OGD-induced protein levels were evaluated by immunoblotting. Cells were transfected with Prx1 plasmid followed by 6 h of OGD or control stimulation. Cell lysates were prepared and resolved by SDS-PAGE. The proteins were immunoblotted with antibodies against phospho-ERK, phospho-JNK, phospho-P38, phospho-FKHR (Ser256), and HO-1. (F) Quantitative analysis of protein levels for (E) was performed by densitometry. Data are expressed as densitometry ratio of control (mean±SEM). **p<0.01 versus control; ^#p<0.05; ^##p<0.01 versus OGD. (G) Changes in apoptosis 6 h after OGD were detected using the TUNEL assay. Double staining was performed for TUNEL (green) and DAPI (blue). The representative images show the increased percentage of TUNEL-positive apoptotic endothelial nuclei (green fluorescence) 6 h after OGD. Scale bar=100 μm. (H) Quantification of TUNEL-positive apoptotic endothelial cells with or without Prx1 transfection. Apoptosis was dramatically reduced following the overexpression of the Prx1 gene in endothelial cells after OGD. **p<0.01 versus OGD group. (I) Representative flow cytometric dot plots of apoptotic cells after OGD with or without Prx1siRNA transfection. Cultured EA.hy926 cells were stimulated for 6 h with OGD with or without Prx1siRNA transfection. Cells were double-stained with Annexin-V and PI and analyzed by FACS. Immunoblots are representative of three independent experiments. β-Actin was used as the loading control. DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; HO-1, heme oxygenase-1; JNK, c-Jun N-terminal kinase; PARP, poly ADP-ribose polymerase; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PI, propidium iodide. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars