(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.

(A) Inhibition of Stat3 decreases RelA acetylation in A2058 cells. siRNA transfected cells were immunoprecipitated with anti-RelA antibody; endogenously acetylated RelA protein (Ac-RelA) was then detected by anti-Ac lysine antibody (left panel). Stat3 inhibition by siRNA is shown in the right panel. (B) Constitutively active Stat3C enhances TSA-mediated RelA acetylation while inhibiting interaction between RelA and IκBα. BALB/c 3T3 cells were transfected with indicated expression vectors and treated (18 h) with TSA. Western blot analyses with specified antibodies are shown. (C) Stat3 activity inhibits tumor nuclear RelA’s affinity to IκBα. Nuclear RelA from A2058 tumor cells treated with control or Stat3 siRNA was allowed to interact with recombinant IκBα, followed by Western blot analysis. The effects of Stat3 siRNA are shown at the bottom. (D) Stat3 activators frequently elevated in tumor, such as IL-10, induce acetylation of RelA in primary immune cells in a Stat3-dependent manner. Splenic DCs isolated from Stat3^+/+ or Stat3^−/−were treated with either TNFα or IL-10, followed by Western blot analyses.

(A) Stat3 Ser727, Y705 and Stat3 DNA binding are all important for RelA acetylation. Stat3-deficient MEFs were transfected with Stat3 WT, or Stat3 S727A or Y705, Stat3D (dominant-negative DNA-binding mutant) or Stat3C (constitutively activated mutant). Samples shown in the left panels were treated with OSM to stimulate Stat3 activation. (B). RelA acetylation is critical for Stat3-mediated RelA nuclear retention. Left panel: Stat3C and p300 were co-transfected with WT RelA, or RelAK221R, K310R or K218/221/310R. RelA levels in nuclear and cytoplasmic extracts were determined by Western blot. Co-immunoprecipitation to determine RelA acetylation was performed using the nuclear extract. Right panel: OSM was used to stimulate Stat3 in Stat3^−/− MEFs transfected with indicated vectors. Nuclear and acetylation levels of RelA were detected as described above.

RelA is constitutively acetylated (A) and phosphorylated (B) in human tumors and colocalizes with p-Stat3. Sections of human lung cancer specimens were stained with the indicated antibodies, followed by confocal microscopic analyses. Lower panels are enlarged images of the top panels. Top panels scale bar = 40μm; bottom panels scale bar = 10μm.

(A) Cellular localization of p-Stat3 (green) and p-RelA (red) was observed by confocal microscopy after immunofluorescent staining of A2058 cancer cells treated with Stat3 or control siRNAs. Photos with higher magnification were shown in the upper panels. Scale = 20μm. (B) Stat3-dependent p-RelA accumulation in tumor cells. Left: Western blot analyses of nuclear and cytoplasmic p-RelA in indicated cancer cells transfected with either control or Stat3 siRNAs. HDAC1 and tubulin proteins were used as nuclear and cytoplasmic controls, respectively. Middle panel: Physical interaction of p-RelA with p-Stat3 in tumors. Nuclear extracts from A2058 tumor cells were immunoprecipitated using anti-RelA antibody or anti-Stat3 or pre-immune serum (PIS) control, followed by immunoblotting analysis with specified antibodies. Right panel: Effects of knocking down Stat3 and RelA on p-RelA vs. total RelA in the nucleus.

(A). Stat3-associated RelA acetylation is mediated by p300. Left: Activated Stat3 increases p300-mediated RelA acetylation. 3T3 cells were transiently transfected with plasmid expressing p300 and/or Stat3C together with RelA, followed by Western blotting. Right: p300 protein interacted with acetylated RelA in a Stat3-dependent manner. B16 melanoma cells were transfected with either control or Stat3 siRNA. Nuclear RelA proteins were immunoprecipitated with anti-RelA antibody followed by Western blot analysis with indicated antibody. Total p300 levels were determined in whole cell extracts (bottom panel). (B). Tumor NF-κB-DNA complex contains both p300 and Stat3, whereas TNFα-induced NF-κB-DNA complex in DCs does not. Top left: Nuclear extracts prepared from B16 tumors were subjected to EMSA to detect NF-κB DNA-binding activity. Specific antibodies as indicated were used to identify Stat3, RelA and p300 in the binding complex. Top right: Nuclear extracts from B16 tumors were incubated with biotin-labeled oligonucleotides containing NF-κB sites and the DNA-bound RelA complex was pulled down by Streptavindin beads, followed by Western blotting. Bottom left: NF-κB RelA/p50 complexes with p300 and Stat3 in A2058 cancer cells. Bottom right: NF-κB-DNA complexes in DC2.4 cells after TNFα treatment was distinct from that found in tumor cells with constitutively-activated Stat3 (bottom, right).

(A) CD11b+ myeloid cells were purified from B16 tumors harvested from mice with Stat3^+/+ and Stat3^−/− myeloid compartment, followed by Western blot analysis utilizing an antibody specific for Ac-K310 of RelA. (B) High levels of acetylated RelA in B16 tumor cell nuclei from mice with Stat3^+/+ myeloid cells but not in tumors grown in mice with Stat3^−/− myeloid cells. Frozen sections from OCT embedded tumor tissues were stained with an antibody against acetylated RelA (green) and mounted in medium containing DAPI to show nucleus (blue). Middle panels are enlarged images of the left panels. Left and right panels scale bar = 200μm; middle panels scale bar = 50μm.