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. 2011 Aug;31(16):3457–3471. doi: 10.1128/MCB.05523-11

Fig. 3.

Fig. 3.

Foxo1 directly regulates MMP1 and mediates the connection between Cdc25A and MMP1. (A) Modulation of Foxo1 impacts MMP1 protein levels. The plasmid Foxo1-WT-RE or Foxo1-S249A-RE was transfected into the indicated cells with Foxo1 depletion via Foxo1-siRNA. The cell lysates were immunoblotted with the indicated antibodies. (B and C) The mutation Foxo1-S249A blocks the effects of Foxo1 on MMP1. The human Cdc25A and MMP1 mRNA levels were evaluated in the cells with Foxo1-WT-RE (B) and Foxo1-S249A-RE (C) by qRT-PCR (mean ± SD; n=5). The Cdk2 and Foxo1 mRNA levels served as loading controls. (D) The conserved putative Foxo1 binding sites in the MMP1 promoter (http://www.ifti.org/cgi-bin/ifti/Tfsitescan.pl). (E) ChIP analyses of Foxo1 binding in breast cancer cells established binding of Foxo1 to a conserved Foxo1 binding motif at 572 bp upstream of the MMP1 transcription start site. The results are presented as fold template enrichment in immunoprecipitates of Foxo1 antibody relative to those of a control antibody (mean ± SD; n=5). (F) Mutated derivatives of the pGL4-MMP1 promoter with three putative Foxo1 binding sites. (G) pGL4-MMP1-promoter or the mutated derivatives pGL4-MMP1-promoter-mut (Mut1, -2, or -3) was transfected into breast cancer cells with Foxo1-WT or Foxo1-siRNA and then assayed for its activity. The empty vectors served as controls (mean ± SD; n=5).