Fig. 6.

Inhibitory effect of EA/EuH on TNFα-induced NF-κB activation. a Serum-starved MDA-MB-231 cells were pre-treated with EA/EuH (5 μg/mL) for 30 min before stimulation with TNFα (10 ng/mL). After 20 min, whole-cell lysates were prepared, and immunoblotting was performed using the phospho-specific antibody against IκBα (Ser32) or RelA/p65 (Ser536) as indicated. The anti-GAPDH antibody was used as an internal control. b MDA-MB-231 cells were pre-treated with EA/EuH (5 μg/mL) for 30 min before stimulation with TNFα (10 ng/mL). After 20 min, the cells were fixed and incubated with antibodies against α-tubulin or phospho-RelA/p65 (Ser536) for 2 h, followed by incubation with Alexa Fluor 488-conjugated (green signal) or Alexa Fluor 555-conjugated (red signal) secondary antibody for 30 min. Nuclear DNA was stained with 1 μg/mL Hoechst 33258 for 10 min (blue signal). c MDA-MB-231 cells were transfected with 5× NFκB-Luc plasmid along with 50 ng pRL-null. At 48 h post-transfection, the cells were treated with or without 10 ng/mL TNFα in the absence or presence of EA/EuH (5 μg/mL). The data shown represent the mean ± SD. ***, P < 0.0001 (n = 9). P value was analyzed by Sidak’s multiple comparisons test