Fig. 2: p53 stabilization induces nuclear deformation, Lamin A/C expression, and p16 expression.

a Nutlin-3 induces nuclear deformation in parental HCT116 (HCT p53+/−) cells in a time-dependent manner (left). Nuclear deformation rate by Nutlin-3 was counted (left). HCT p53+/− cells were treated with Nutlin-3 (1 μM) for different time (24, 48 h). After treatment, cells were IF stained with Lamin A/C (Red) and counterstained with DAPI (Blue). *P < 0.05, **P < 0.03, NS, not significant. b Western blotting image shows that stabilized p53 by Nutlin-3 treatment increases expression of Lamin A/C, p16, and NOXA. HCT p53+/− cells were treated with Nutlin-3 (1 μM) for different time period (3–48 h). After treatment, cell extracts were analyzed by western blotting. Actin was used as loading control. Western blotting data of three independent experiments are shown. Lower and weak bands in Lamin A/C blot are Lamin C (LC). c Immunofluorescence images of nuclear deformation and p16 expression after Nutlin-3 treatment for different time period. HCT p53+/− cells were treated with Nutlin-3 (1 μM) for different time (24, 48 h). After treatment, cells were subjected to immunofluorescence staining for Lamin A/C (Red), p16 (Green), and counterstained with DAPI (Blue). d Immunofluorescence images of nuclear deformation and decreased expression of H3K9me3 in HCT p53+/− cells (left). Counting of histone H3K9me3-positive cells (middle) and signal intensities (right) on the basis of IF staining. HCT p53+/− cells were treated with Nutlin-3 (1 μM) for different time (24, 48 h). After treatment, cells were IF stained with Lamin A/C (Red), H3K9me3 (Green), and counterstained with DAPI (Blue). *P < 0.05. e p53 stabilization increases cellular senescence. SA-β-gal staining shows Nutlin-3 treatment increased senescence in p53-positive cell (left) in a time-dependent manner. Counting of β-gal-positive cells (right). HCT p53+/− cells were treated with Nutlin-3(1 μM) for different time (24, 48 h). After treatment, cells were stained with SA-β-gal. Boxes indicate magnified regions displayed in the right panel. *P < 0.03. f p53 stabilization decreases cell proliferation. Ki-67 staining is reduced after p53 stabilization for accompanying cells with nuclear deformation (left). Counting of Ki-67-positive cell (middle) and signal intensity (right) on the basis of IF staining. HCT p53+/− cells were treated with Nutlin-3 (1 μM) for different time (24, 48 h) followed by IF staining using Lamin A/C (Red), Ki-67 (Green), and counterstaining using DAPI (Blue). *P < 0.05, **P < 0.03