Mitotic arrest after NEDD1-depletion depends on the checkpoint protein p53 in HCT116 cells. (A) Flow-cytometric analysis of HCT116 p53+/+ and HCT116 p53 -/- cells treated with control RNA or siRNA targeting NEDD1 at 50 nM for 72 h and double-labelled with propidium iodide and FITC-conjugated anti-cyclinB1 antibody. (B) Quantification of cyclinB1-positive cells (error bars represent SD from three independent experiments). (C) Immunofluorescence of mitotic HCT116 p53+/+ and p53 -/- cells, 72 h after transfection with control or NEDD1-siRNA, using anti-alpha tubulin and DAPI staining. Bar, 20 μm. (D) Percentage of mitotic cells shown in (C). 500 cells were scored per condition (error bars represent SD from three independent experiments). (E) Immunoblots of HCT116 p53+/+ and p53 -/- cells treated 48 h with control or NEDD1-siRNA. Blots were probed with antibodies against phospho-histone H3 and actin.