Figure 2.

IRF1 is indispensable for BV6-mediated TNFα induction and selectively controls BV6-induced NF-κB target genes. MDA-MB-231 cells were transiently transfected with 5 nM siRNA against IRF1 or control siRNA. (a) Protein levels of IRF1 were analyzed after 24 h by western blotting. β-Actin served as a loading control. (b) Cells were treated with 50 nM BV6 for indicated time points. TNFα mRNA levels were assessed by quantitative reverse transcriptase-PCR (qRT-PCR) and normalized to 28S rRNA expression (b). Data are presented as fold increase of untreated control siRNA cells. Mean±S.E.M. of two independent experiments performed in duplicate are shown. (c) Cells were treated with 50 nM BV6 for 15 h, cell culture supernatants were harvested and assessed for TNFα protein levels by ELISA. Data are presented as fold change of treated control siRNA cells. Mean±S.D. of three independent experiments performed in duplicate are shown. (d) Cells were treated with 50 nM BV6 for 15 h. TNFα of 50 pg/ml was additionally added after 15 h and cells were analyzed after 48 h in total. Apoptosis was determined by DNA fragmentation of PI-stained nuclei using flow cytometry and the percentage of DNA fragmentation is shown with mean±S.D. of three independent experiments performed in duplicate. (e) MDA-MB-231 cells stably expressing IκBα-SR or vector control were treated for indicated time points with 50 nM BV6. IL-8, p100 and RelB mRNA levels were assessed by qRT-PCR and normalized to 28S rRNA expression. Data are presented as fold increase of untreated vector control cells. Mean±S.E.M. of two independent experiments performed in duplicate are shown. (f) MDA-MB-231 cells were transiently transfected with 5 nM siRNA against IRF1 or control siRNA and were treated for indicated time points with 50 nM BV6. IL-8, p100 and RelB mRNA levels were assessed by qRT-PCR and normalized to 28S rRNA expression. Data are presented as fold increase of untreated control siRNA cells. Mean±S.E.M. of three independent experiments performed in duplicate are shown. (g) MDA-MB-231 cells stably expressing a GFP-labeled NF-κB reporter construct were transiently transfected with 5 nM siRNA against IRF1, p65 or control siRNA and treated for 15 h with 50 nM BV6. NF-κB transcriptional activity was assessed by flow cytometry and is displayed as fold increase of untreated control siRNA cells. Mean±S.D. of three independent experiments performed in duplicate are shown. *P<0.05; **P<0.01