Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2008 May 23.

Published in final edited form as: Nat Cell Biol. 2007 Mar 18;9(4):470–478. doi: 10.1038/ncb1559

(a) Hs578T cells were transiently transfected, in triplicate, with 0.5 μg p1.7 RELB promoter-Luc vector, 0.5 μg of the indicated AP-1 subunit expression vector, 0.5 μg SV40-β-gal and empty vector (EV) pcDNA3 to make a total of 3.0 μg DNA. Post-transfection (48 h), luciferase and β-gal activities were determined and normalized luciferase activities presented (mean ± S.D. from three separate experiments). (b) The indicated breast cancer cell lines were transiently transfected with 1.0 μg p1.7 RELB promoter-Luc vector, 0.5 μg c-Jun and Fra-2 AP-1 subunit expression vector, 0.5 μg SV40-β-gal and EV DNA to a 3.0 μg DNA total, and analyzed as in (a). (c) Hs578T or MCF-7 cells were transiently transfected with 0.5 μg p1.7 RELB promoter-Luc DNA, 0.5 μg of the indicated AP-1 subunit expression vector, in the absence or presence of 0.5 μg IκB-α vector DNA, 0.5 μg SV40-β-gal and EV DNA to a 3.0 μg DNA total, and processed as above. (d) Hs578T cells were transiently transfected with 0.5 μg of WT or κB-mutant p1.7 RELB promoter-Luc vector, AP-1 subunit, SV40-β-gal and EV DNA and analyzed as in (a). (e) MDA-MB-231 or Hs578T cells were transfected with siRNA directed against JUN or FRA2, or against both genes. All duplexes were used at a concentration of 100 nM and 100 nM GFP siRNA was added when only one gene was targeted. Post-transfection (48 h), WCEs were analyzed by immunoblotting for RelB, c-Jun, Fra-2, and β-actin. (Please note loading for si-JUN and si-FRA2 was reversed in the two blots.) The blots were scanned and the values for RelB, c-Jun, and Fra-2 normalized to β-actin. Values relative to the control siRNA (set at 100%) are given below each lane, showing their corresponding levels of decrease. (f) The indicated breast cancer cell lines were transiently transfected with 0.5 μg p1.7 RELB promoter-Luc vector, 0.25 μg of the indicated AP-1 or NF-κB subunit expression vectors, 0.5 μg SV40-β-gal and EV DNA to a 3.0 μg DNA total, and analyzed as in (a).

(a) Hs578T cells were transiently transfected, in triplicate, with 0.5 μg p1.7 RELB promoter-Luc vector, 0.5 μg of the indicated AP-1 subunit expression vector, 0.5 μg SV40-β-gal and empty vector (EV) pcDNA3 to make a total of 3.0 μg DNA. Post-transfection (48 h), luciferase and β-gal activities were determined and normalized luciferase activities presented (mean ± S.D. from three separate experiments). (b) The indicated breast cancer cell lines were transiently transfected with 1.0 μg p1.7 RELB promoter-Luc vector, 0.5 μg c-Jun and Fra-2 AP-1 subunit expression vector, 0.5 μg SV40-β-gal and EV DNA to a 3.0 μg DNA total, and analyzed as in (a). (c) Hs578T or MCF-7 cells were transiently transfected with 0.5 μg p1.7 RELB promoter-Luc DNA, 0.5 μg of the indicated AP-1 subunit expression vector, in the absence or presence of 0.5 μg IκB-α vector DNA, 0.5 μg SV40-β-gal and EV DNA to a 3.0 μg DNA total, and processed as above. (d) Hs578T cells were transiently transfected with 0.5 μg of WT or κB-mutant p1.7 RELB promoter-Luc vector, AP-1 subunit, SV40-β-gal and EV DNA and analyzed as in (a). (e) MDA-MB-231 or Hs578T cells were transfected with siRNA directed against JUN or FRA2, or against both genes. All duplexes were used at a concentration of 100 nM and 100 nM GFP siRNA was added when only one gene was targeted. Post-transfection (48 h), WCEs were analyzed by immunoblotting for RelB, c-Jun, Fra-2, and β-actin. (Please note loading for si-JUN and si-FRA2 was reversed in the two blots.) The blots were scanned and the values for RelB, c-Jun, and Fra-2 normalized to β-actin. Values relative to the control siRNA (set at 100%) are given below each lane, showing their corresponding levels of decrease. (f) The indicated breast cancer cell lines were transiently transfected with 0.5 μg p1.7 RELB promoter-Luc vector, 0.25 μg of the indicated AP-1 or NF-κB subunit expression vectors, 0.5 μg SV40-β-gal and EV DNA to a 3.0 μg DNA total, and analyzed as in (a).