(Calbiochem). Images were collected by confocal laser microscopy (model TCS-SP2-AOBS, Leica, Germany). SA ß-Gal activity Senescence-associated (SA) ß-galactosidase (ß-Gal) activity was measured as previously described [45], except that cells were incubated in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (XGal) at pH 5.5 to increase the sensitivity of the assay in MEFs. The percentage of cells expressing SA ß-Gal was quantified by inspecting .400 cells per 10-cm-diameter plate three times. Protein phosphatase assays PP1 activity was determined according to standard procedures as previously described [57]. PP activity was assayed using 32P- labeled phosphorylase a as a substrate which detects both PP1 and PP2A activities. To selectively measure PP1 activity we used 2 nM okadaic acid to selectively inhibit PP2A. The cell pellet was homogenized in the extraction buffer containing 20 mM Tris- HCI, pH 7.5, 5 mM EDTA, 10 mM EGTA, 15 mM -mercap- toethanol, 0.25 M sucrose, 0.3% Triton X-100, 5 mg/ml leupep- tin, and 5 mg/ml aprotinin and centrifuged to give a soluble supernatant. The PP activity in the clear supernatant was determined by measuring the trichloroacetic acid-soluble counts released after incubation of the 32P-labeled phosphorylase a in the cell extract. The PP activity was linear up to assay times of 10 min and 5 mg protein of the cell extract. Routinely, incubation for PP activity was carried out for 10 min with an extract containing 5 mg of protein as determined by the Bio-Rad assay (Bio-Rad, Hercules, CA). Negative controls were obtained incubating with 100 nM Okadaic acid to inhibit PP1 and PP2A activity. One unit (U) of activity is defined as the amount that catalyzes the release of 1 nmol Pi from phosphorylase a per min at 30uC. 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