Figure 1 - Identification of a small molecule that reverses senescence.

(a) Dominant-negative TRF2 expression construct used to elicit cellular senescence. (b) Experimental outline and reference time frame. (c) Live-cell imaging–based high-throughput screen for small molecules that reverse the senescent phenotype elicited by TRF2^BM. BJ cells were induced to enter into senescence by infection with the retrovirus depicted in a, plated in 96-well plates and treated with the compounds in a chemical library at a concentration of 1 g ml^-1. Live-cell images of EGFP-positive cells were captured by automated fluorescence microscopy at the indicated times. Enlarged views of the images taken 1 d after compound treatment are also shown; cells escaping senescence and cells still in a senescent state are marked with red and yellow arrowheads, respectively. (d,e) Quantitative analysis of the effects of CGK733 on the growth (d) and mean size (e) of cells with TRF2^BM-induced senescence. (f) Reversible changes in cell morphology and SA–-gal expression by CGK733 in cells with TRF2^BM-induced senescence. In c–f, CGK733 was provided at 1 g ml^-1. Scale bars, 200 m (c,f). (g) Growth response of cells with of TRF2^BM-induced senescence to various doses of CGK733. The increase in cell number over 6 d after CGK733 treatment is shown as a ratio. Average of three experiments are shown; error bars, s.d. (h) Reversible manipulation of cellular lifespan by CGK733. BJ cells were cultured until replicative senescence and were serially passaged with or without 2 M CGK733. Red and blue arrowheads indicate the times of addition and withdrawal of CGK733, respectively. The asterisk indicates the PD level at which the cell population was almost completely senescent.