Figure 1: Effect of DMBA carcinogen in primary MEFs. A) Growthcurve of primary cells (MEFs) treated DMBA alone (DMBA, ), oruntreated (). Arrow indicates the time of treatment with DMBA. B)FACS analysis of the proliferative status of the cells treated withDMBA (bottom figure), or vehicle only (upper figure). C) 104MEFs were seeded in a 10 cm plate and treated with 10 M DMBA.After 7-10 days the cells were fixed and stained with crystal violet toidentify clones. Arows indicate actively growing clones. D)Phenotype of cells treated with DMBA. E) Individual clones weresubcultivated independently and the presence of p53 was analyzedby Western blot. Cells expressing mutant p53 (175H) were used ascontrol. F) Picture of a representative actively growing clone fromC. Arrows indicate senescent cells. G) Quantification of thepercentage of senescent cells from 3 independent experiments.Senescent cells were processed as indicated in materials andmethods and more than 400 cells counted in each experiment. Datashows the average of the senescent cells. Barrs show SD. *: p<0.05,**: p<0.01, ***: p<0.001, Student’s T test.