Figure 6

FOXM1 regulates ATM phosphorylation and NBS1 expression through a FHRE site within its promoter. (a) MCF-7 cells were transfected with pcDNA3 empty vector or pcDNA3-FOXM1 plasmids following which the cells were subjected to 1 μM epirubicin treatment for 0, 6 24 and 48 h. Western blot analyses were performed to analyse the protein expression level changes of FOXM1, NBS1, P-ATM, ATM, RAD50, MRE11 and β-tubulin. (b) MCF7-Epi^R cells were transfected with NS siRNA or NBS1 siRNA. Twenty-four hours after the transfection, cells were treated with 1 μM epirubicin for 0, 4, 24 and 48 h and harvested for western blot analyses. Protein expression levels of the indicated proteins: NBS1, P-NBS1, FOXM1, ATM, P-ATM, RAD50, MRE11, PARP and β-tubulin were analysed (arrow indicates cleaved PARP proteins). (c) MCF-7 cells were transciently transfected with 20 ng of pGL3-NBS1 WT or pGL3-NBS1 mut promoter together with the control Renilla plasmid and increasing amounts (0, 10, 15 and 20 ng) of FOXM1 expression vector. After 24 h, cells were assayed for luciferase activity. The relative luciferase activity was calculated after normalizing with the control Renilla activity. (d) MCF-7 either untransfected or transfected with pcDNA3-FOXM1, and MCF7-Epi^R either untreated or treated with 1 μM epirubicin for 16 h were used for chromatin immunoprecipitation assays using the IgG as negative control and anti-FOXM1 antibody, as indicated. After reversal of cross-linking, the coimmunoprecipitated DNA was amplified by PCR, using primers amplifying the FOXM1 FHRE-binding site containing region (−300/−24 bp) and a control region (−1097/−826 pb), and resolved in 2% agarose gel. Inverted ethidium bromide stained images are shown.