FIG 1.

Premature senescence in HGPS cells is dependent on p53 and not dependent on telomere shortening. (A) Measurement of the number of mean population doublings over the life span of normal AG08 fibroblasts (left) and HGPS AG11 fibroblasts (right) expressing p53 shRNA, hTERT, or p53shRNA plus hTERT. Data represent averages from 3 biological replicates counted with 4 technical replicates each week. Standard error of the mean (SEM) values are shown as error bars. (B) β-Galactosidase staining of normal AG08 and HGPS AG11 fibroblasts expressing hTERT alone or p53shRNA plus hTERT at the indicated mean population doubling (MPD). (C) The G₁/S ratios were determined from cell cycle profiles obtained by flow cytometry after propidium iodide staining. Cellular senescence is reflected by an increase in the G₁/S ratio. (D) TRF assay of AG08 and AG11 cells showing overall telomere length in young (Y), senescent (S), and hTERT (hT)-expressing cells. This assay was repeated 3 times, and a representative result is shown. The average telomere length, determined by pixel density maxima, is shown. (E) Western blot analysis of young cycling and senescent AG08 and AG11 cells; AGO8 and AG11 cells expressing hTERT at population doubling 35 were also compared. Protein extracts were immunoblotted with the indicated antibodies. HCT116 lysates served as a positive control for p16 expression.