Fig. 3

Sac-1004 suppresses IL-1β-induced NF-kB activation. HBMECs were allowed to grow to confluence. Starved cells were treated with Sac-1004 (10 μg/ml, 1 h) followed by IL-1β treatment. Western blotting was performed using anti-p-IκB and anti-NF-κB antibodies. Cells were then fixed, permeabilized, and subsequently immunostained for NF-κB. Translocation of the NF-κB protein to the nucleus and cytosol fractions was observed. IL-1β (10 ng/ml, 6 h) induced the translocation of NF-κB to the nucleus, but in the presence of Sac-1004, the translocation of NF-κB was reduced (a, b). Sac-1004 reduced the IL-1β (10 μg/ml, 30 min)-induced expression level of p-IκB (c). HBMECs were transfected with a NF-κB p65 reporter construct. The next day, the transfected cells were treated with Sac-1004 (10 μg/ml, 1 h) followed by IL-1β (10 ng/ml, 12 h) treatment. Luciferase activity from the NF-κB p65 reporter constructs was measured. The luciferase reporter assay also showed a similar reduction in IL-1β-induced NF-κB activation after treatment with Sac-1004 (d). Data are shown as relative activity compared with a mock vector. Transfection efficiency was normalized to Renilla luciferase activity from co-transfected pRL-CMV. All data are presented as means ± SEM. *P < 0.05