Figure 3.

Ectopic expression of FOXM1 suppresses the mitotic block caused by loss of p53 in human epidermal keratinocytes. (a–d) Analyses 3 days after infections with CT1 or shp53-1 and CT2 or FOX (as in Figure 1). (a) Expression of cell cycle regulators p-Rb, Cyclin E (CycE), Cyclin A (CycA) and Cyclin B (CycB) by western blotting. GAPDH (GAP) was used as loading control. (b) Percent of cells in the G1 (2N), S, G2/M (4N) or polyploid (>4N) phases of the cell cycle as determined by flow cytometry. More details in Supplementary Figures 2a and b. (c) Percent of BrdU-positive cells in the G1, S (2N), G2/M (4N) and polyploid (>4N) phases as in (b). More details in Supplementary Figure 2c. (d) Binucleated keratinocytes (white arrows) bearing shp53 and CT2 or FOX as indicated, stained with DAPI for DNA. White numbers are the percent of polyploid cells (nuclei >15 μm) that were binucleated. (e) Immunodetection of endogenous FOX (green), CycA (red, left panel) and CycB (red, right panel) in normal human epidermis by immunofluorescence. DAPI for DNA in blue. Red line is nonspecific staining of the superficial cornified layer. Arrows for cells coexpressing FOX and CycA or CycB. Broken line for the basement membrane. (f) Detection of nascent transcription by 5′-fluorouridine 5′-Flu; green) incorporation 2 days after infections as indicated (nucleolar foci are indicated on representative photographs by white arrows). Dot plot on the left side represents quantitation of the fluorescence intensity of 5′-Flu incorporating single foci. Black bars represent the mean. Scale bars: 50 μm; *P<0.05 and **P<0.01. Data are mean±s.e.m. of triplicate samples of representative experiment (n=3).