Fig. 2.

Nox-4 induces nuclear c-Jun DNA binding and AP-1 transcriptional activation. Successful knockdown of nox-4 after siRNA transfection was confirmed by real-time PCR (mRNA) (A) and Western blot (protein) (B). DNA binding activity (C) of c-Jun from EOMA cell nuclear protein extracts was performed using an ELISA-based (Trans-AM) method. There was significant decrease in c-Jun DNA binding activity with Nox-4 knockdown. D: EOMA cells were transfected with Nox-4 siRNA; after 72-h transfection, cells were washed and cotransfected with a pAP-1 luciferase and pRL-TK Renilla luciferase plasmids for 48 h. Firefly luciferase (FL) activity levels were normalized with Renilla luciferase(RL). Nox-4 knockdown in EOMA cells resulted in loss of AP-1 transcriptional activation. E: confocal images of immunofluorescence detection of phospho-c-Jun in murine and human EC tumor sections with human AVM and normal blood vessel in human skin as EC controls. F: quantification of fluorescence signal intensity using Olympus FV10-ASW software. Results are expressed as means ± SD. *P < 0.05; for tumor sections, n = 5 fields per section.