Fig 8. p27^Kip1 S140 phosphorylation is required for survival after DNA damage.

(A) U20S cells expressing RFP or GFP were transfected with control siRNA or a p27-specific siRNA, respectively. The next day the GFP/p27siRNA cells were transfected with vector alone, myc-p27WT, myc-p27S140A or myc-p27S140D and the RFP/control siRNA were transfected with vector alone only. Two days later the cells were harvested and a portion was analyzed by Western blotting to assess construct expression with the indicated antibodies (GFP cells only). (B) The rest of the cells were analyzed in a multicolor competition assay by flow cytometry. The relative survival of the control siRNA-treated cells was set to 100%. Data is presented as mean of 3 independent experiments ± SEM. Differences between groups were evaluated using two-tailed Student t tests among replicate experiments; *P = 0.0249. (C) U2OS cells were transfected with vector alone, myc-p27WT or myc-p27S140A expressing constructs. Two days later the cells were subjected to 0 (-IR) or 6 Gy of IR (+IR), fixed 1h later and the localization of p27WT and p27S140A was determined by immunofluorescence microscopy with a myc antibody and DAPI staining.