Crosstalk between NFκB and GLI1 signaling pathways. (A) Immunofluorescence images showing localization of NFκB p50 (Panel I) or p65 (Panel II) subunits in HMLE-shEcad (top row) or HMLE-shGFP (lower row) cells. Scale bar indicates 50 μm. (B) Map of putative NFκB binding sites surrounding the GLI1 promoter. Red arrow marks transcription start site. Green lines indicate sequences matching the consensus κB binding site; putative sites are numbered 1 to 6. Blue arrows indicate primer sets used for ChIP. (C) ChIP of HMLE-shEcad cells with indicated antibodies. Error bars indicate standard error among three independent experiments. (D) NFκB p65 ChIP to the GLI1 promoter (site 1) following six-hour treatment with 1 μM triptolide. Graph was normalized to binding with vehicle control (DMSO). Error bar indicates standard error among three independent experiments. (E) Real-time PCR data showing RELA, NFKB1, and GLI1 transcript levels after infection of HMLE-shEcad cells with non-targeting (NT) or shRELA and shNFKB1 virus (shNFκB) virus. (F) Western blot of Gli1, p65, and p50 levels in HMLE-shEcad cells infected with either non-targeting (NT) or shRELA and shNFKB1 virus (shNFκB). GAPDH serves as a loading control. For all: * = P ≤0.05, t test. ** = P ≤0.005, t test. ChIP, chromatin immunoprecipitation; GLI1, glioma-associated oncogene 1; RELA, v-rel reticuloendotheliosis viral oncogene homolog A; NFKB1, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; sh, short hairpin.