Figure 1. FOXO4 induces cellular senescence in endogenous BRAF^V600E-expressing Colo829 and A375 cells.

a) Ectopic FOXO4 expression reduces proliferation of Colo829 cells. Colo829 cells transiently expressing HA-FOXO4 were subcultured in puromycin containing selection medium and stained for colony outgrowth. Additionally, a set of cells were stained at 2.5 days post transfection with anti-PCNA or analyzed for BrdU incorporation. 250 non-transfected and 50 transfected cells were quantified. Similar results were obtained in A375 melanoma cells. EV=Empty vector. b) Ectopic FOXO4 expression induces SA-β-GAL positivity in Colo829 cells. Colo829 cells expressing HA-FOXO4 were selected with puromycin and stained for colony formation, or SA-β-GAL. Protein samples were obtained at 2.5 days post transfection and analyzed by immunoblotting. 50 colonies were quantified for positive cells. c) FOXO4 expression in Colo829 cells induces senescence. Colo829 cells were transfected as in a) and at 5.5 days post transfection stained with DAPI to visualize Senescence Associated Heterochromatin Foci (SAHF) formation in parallel with anti-H3K9-Me(III) for H3K9-trimethylation. 100 cells were quantified and the percentage of double positive cells indicated.