Figure 1. KEAP1 selectively inhibits the NF-κB signaling pathway.

(A) Ectopic of KEAP1 suppressed TNFα–mediated RELA nuclear translocation. Hs578T breast cancer cells were transfected with either RFP-KEAP1 or vector, serum-starved overnight, treated with 2 ng/ml TNFα for 30 min, stained with anti-RELA antibodies (green), and examined by confocal microscopy. The nucleus was stained with DAPI (blue). The arrow indicates KEAP1-expressed cells (red).

(B) Depletion of endogenous KEAP1 by KEAP1 siRNA led to accumulation of nuclear RELA.

(C) Knockdown of KEAP1 increased TNFα–induced NF-κB activation. The 5IκB-Luc reporter and TK-rLuc (internal control) were transfected with either KEAP1 siRNA or control siRNA into MDA-MB-435 cells. Then, 48 hr post-transfection, cells were serum-starved overnight and treated with 2 ng/ml TNFα. After 8 hr of TNFα treatment, cells were recovered in 1% serum medium overnight and then lysed for luciferase assays. Error bars represent SDs (n=3).

(D) Silencing KEAP1 by KEAP1 siRNA up-regulated the expression of NF-κB–responsive genes in human breast cancer cells. A heat map depicts the relative expression of 12 NF-κB–dependent genes by qRT-PCR. A nonspecific siRNA was used as the control.

(E–F) In vitro angiogenesis assays showed that the silencing of KEAP1 increase HUVEC tube formation and migration in comparison with the control. Depletion of IL-8 by antibodies to IL-8 (α-IL-8) suppressed knockdown of KEAP1-induced HUVEC migration and tube formation. Error bars represent SDs (n=3).

(G–H) Depletion of KEAP1 following TNFα stimulation increased IL-8 mRNA and protein levels as determined by qRT-PCR and ELISA, respectively. Knockdown of NRF2 had no effects on KEAP1 depletion–induced IL-8 expression. Error bars represent SDs (n=3).