NF-κB, AP-1 and C/EBP transcription factors are acting together to control IL-8 gene expression A. MDA-MB-231 cells were infected at MOI 100 with backbone Ad5 or Ad-IκB viruses. 24h after infection, the medium was collected and assayed for IL-8 levels by ELISA. Results represent the mean ± SD of three independent experiments. B. RNA were collected from the same cells and used to measure IL-8 RNA levels by quantitative PCR. Results are expressed as arbitrary units corresponding to the ratio of IL-8 levels normalized by rS9 levels (n=3). C. Reduction of IL-8 expression using RNA interference against Fra-1, Fra-2 or c-Jun. MDA-MB-231 cells were transfected with siFra-1, siFRa-2, sic-Jun or siGFP (as control). The extent of silencing of was determined by western blot using antibodies against Fra-1, Fra-2, c-Jun or β-actin (left panel). Secreted IL-8 levels were measured by ELISA 48h afer transfection. Results represent the mean ± SD of three independent experiments. D. xp2-IL8 reporter was cotransfected along with 100 ng of expression vectors of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPα or C/EBPβ in MCF-7 cells. Results show relative luciferase activities (n=3).