FOXF1 re-expression in breast cancer cells inhibits the CDK2-RB-E2F cascade pathway. A, Top panel: Immunofluorescence (IF) analysis of MCF7 cells, transfected with the GFP or HA-FOXF1 expression plasmid DNA, using anti-HA (green) and anti-phospho-CDK2 (Thr160) (red) antibodies. DNA was labeled with DAPI (blue). Bottom panel: IF of total CDK2 protein levels. B, IF analysis of the same cells as (A) with anti-phospho-RB (Ser807/811) (top panel) and anti-RB (bottom panel) antibodies. C, Flow cytometric analysis of the effect of FOXF1 on levels of phospho-CDK2 and phospho-RB proteins. The FACS histogram data are shown in the top panel. The bar graphs (mean ± SD) from three independent FACS experiments are shown in the bottom panel. The asterisk depicts a statistically significant difference (p < 0.05). D, FOXF1 attenuates E2F1 transcriptional activity. We transfected MCF7 cells with E2F-responsive luciferase reporter DNA and different combinations of expression plasmid DNA as indicated. The luciferase data were converted to fold activation relative to the empty vector control (set as 1). Error bars represent SD of three independent experiments. * p<0.05.