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. Author manuscript; available in PMC: 2010 Apr 7.
Published in final edited form as: Cancer Cell. 2009 Apr 7;15(4):283–293. doi: 10.1016/j.ccr.2009.02.015

Figure 1. Stat3 is Required for Maintaining Constitutive NF-κB Activity in Tumors.

Figure 1

(A) Stat3, but not IKKβ, is required to maintain constitutive NF-κB (RelA) activity in tumors. Left panels, DNA-binding of NF-κB was determined by EMSA using nuclear extracts prepared from B16 tumors grown in mice with Stat3+/+ and Stat3−/− myeloid cells. Phospho-RelA(S536) (p-RelA) and phospho-Stat3 (Y705) (p-Stat3) protein levels in tumors are shown by Western blot (lower panel). Right, DNA binding activity of endogenous NF-κB in A2058 tumor cells transfected with the indicated siRNAs (upper right). Supershifting with an anti-RelA antibody (αRelA) was included in the first lane to verify that the band corresponded to RelA complex. The effects of siRNA treatments are shown in the lower panel. (B) Stat3 is required for maintaining endogenous RelA activity while inhibiting IKK-induced RelA activation in DU145 cancer cells. Cells were transfected with indicated siRNAs and treated (15 min) with TNFα. NF-κB activity was determined by EMSA. (C) TNFα induces RelA phosphorylation through activating IKK/pIκBα, whereas RelA phosphorylation induced by tumor-factors is associated with Stat3 activation. Freshly isolated splenic DCs (from mice with Stat3+/+ and Stat3−/− hematopoietic system) were treated with either TNFα or medium conditioned with tumor supernatant then subjected to Western blotting with indicated antibodies.