Satellite DNA signals markedly distend in senescent fibroblasts. (A) α-Sat (green) and sat II (red) hybridization in early passage culture. Distended satellites are found in a small percentage of nuclei (right; see 3D images and Videos 1 and 2). (B) Most cells in late passage senescent cultures show distended satellites. Note that these cells do not have enlarged nuclei, but contain SAHF (DAPI). Insets show examples of threadlike satellite extensions, some of which extend to and contact the nuclear membrane (indicated by arrows and arrowheads). Inset of boxed region shown in black and white to enhance contrast. (C and D) β-Gal staining confirms that the cycling culture (C) contains few senescent cells and the senescent culture (D) contains many. (E) Quantification showing that senescent cultures contain more cells with distended satellites than cycling cultures. (F–J) Senescence was confirmed by staining for p16 (F), p21 (H), and SAHF (DAPI; G), and quantified (J; error bars are standard error). (K) HeLa cultures do not contain cells with distended satellites.