Figure 5.

HCE suppresses TNFα-triggered NFκB promoter activity but does not affect canonical NFκB activation cascade. (A) Confluent HUVECs were cotransfected with an NFκB reporter vector (firefly luciferase) and a control vector (Renilla luciferase). 24 hours after transfection, cells were pre-treated with HCE (100 or 300 ng/ml) for 30 min. TNFα (10 ng/ml) was applied for 5.5 hrs. A Dual-Luciferase® Reporter Gene assay was used to analyse NFκB-dependent reporter gene expression, which is expressed as ratio of firefly luciferase/Renilla luciferase activity. N = 4; *P ≤ 0.05 versusTNFα. (B) Levels of IκBα and β-actin were assessed by Western blot analysis. One representative out of three independently performed experiments is shown, each. (C) The translocation of NFκB p65 subunit into the nucleus was visualized by immunocytochemistry and fluorescence microscopy. The median nuclear fluorescence intensity was analysed. N = 3; n.s. = not significantly different. (D) Nuclear extracts were prepared from HUVEC lysates and subsequently analysed for their NFκB DNA-binding activity via radioactive gel shift assay followed by densitometric analysis. N = 3; n.s. = not significantly different.