Fig. 5. Endogenous p21 modulates pol foci assembly. (A) HCT116 p21^+/+ and HCT116 p21^–/– cells transfected with GFP-pol were UV irradiated (20 J/m²) when indicated. The sub-nuclear distribution of pol was determined by confocal microscopy. The percentage of cells with GFP-pol foci before and after UV irradiation was determined. In all cases, 150 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. (B) p21^+/+ and p21^–/– cells were subjected to UV irradiation and p21 protein levels determined using specific antibodies. Actin was used as a loading control. (C) Confocal analysis of pol organization in p21^+/+ and p21^–/– cells before (NT) and after UV irradiation. (D) p21^–/– cells were transfected with GFP-pol and p21 or EV when indicated and UV irradiated (20 J/m²). At different times, cells were fixed and the sub-nuclear distribution of pol determined by confocal microscopy. The percentage of cells with GFP-pol foci was determined. In all cases, 150 transfected nuclei were counted. Values are the average and error bars are the standard deviation between equivalent samples in three independent experiments. (E) p21^–/– cells transfected with p21 or EV were UV irradiated and p21 protein levels determined using specific antibodies. Actin was used as a loading control.