Quantitative measurements of mH2A and HP1β protein levels in human fibroblasts at different replicative ages. (A) Immunofluorescent images (illustrated in Fig. 1) were quantified as indicated in Experimental procedures and plotted as histograms of cell number (% of total) against fluorescence intensity in arbitrary units (a.u.). mH2A, left panel; HP1β, right panel. Replicative ages of the cultures were as indicated in Fig. 1. A minimum of 100 cells were scored for each age group. (B) Cells were co-stained with mH2A and HP1β antibodies, images were acquired, and individual cells were scored in three channels (using the DAPI, rhodamine and Cy5 filter sets for the nuclei, HP1β and the mH2A, respectively). Data are plotted as 2–D scatter plots of mH2A and HP1β intensities.

Quantitative measurements of mH2A protein levels in mouse lung. (A) Representative images of sections from 4 and 24 month old female lung. The bright foci in the DAPI images are the prominent pericentromeric heterochromatin found in this species. (B) Immunofluorescent images collected in the experiment shown in panel A were quantified as indicated in Experimental procedures, expressed as ratios of mH2A and DAPI intensities for each nucleus, and plotted as histograms of cell number (% of total) against fluorescence intensity in arbitrary units (a.u.). Representative histograms for 2 young (blue) and 2 old (red) animals are shown. (C) Immunoblotting analysis of total tissue extracts from 5 and 36 month old male lungs. Five young animals (lanes 1–5) and 5 old animals (lanes 6–10) were included in the analysis. Lanes were loaded for equivalent DNA content and blots were probed with mH2A and H2B antibodies. (D) Quantification and comparison of mH2A levels detected by immunofluorescence and immunoblotting. Left panel: average intensities of each histogram from the experiment shown in panel B were computed, and means were calculated for the young (blue) and old (red) age groups. A total of 4 young and 6 old animals were included in the analysis. The mH2A level for the young age group was set to 1. Error bars indicate the SEM. The difference in mH2A levels between the young and old groups is significant at p = 0.0094 calculated using a two-tailed student's test. Right panel: immunoblots from the experiment shown in panel C were quantified using the LI-COR Odyssey infrared imaging system. mH2A levels were normalized to H2B levels detected in the same lane. A total of 5 young and 5 old animals were included in the analysis. The mH2A level for the young age group was set to 1. Error bars indicate the SEM. The difference in mH2A levels between the young and old groups is significant at p = 0.0086 calculated using a two-tailed student's test. All experiments were repeated at least twice with consistent results.

Quantitative single cell measurements of mH2A protein levels in mouse skeletal muscle. (A) Representative images of sections from 5 and 36 month old male quadriceps muscle. The bright foci in the DAPI images are the prominent pericentromeric heterochromatin found in this species. (B) Immunofluorescent images collected in the experiment shown in panel A were quantified as indicated in Experimental procedures, expressed as ratios of mH2A and DAPI intensities for each nucleus, and plotted as histograms of cell number (% of total) against fluorescence intensity in arbitrary units (a.u.). Representative histograms for 2 young (blue) and 2 old (red) animals are shown. Note the prominent biphasic profile of the histograms. (C) Quantification of mH2A levels detected by immunofluorescence. The number of cells (% of total) under the low (green) and high (yellow) intensity peaks were calculated for each histogram, and means were calculated for the young and old age groups. A total of 5 young and 5 old animals were included in the analysis. Error bars indicate the SEM. The ratio of low to high intensity cells was 1.48 for the young animals, and 0.99 for the old animals. The difference between the ratios of low and high intensity cells is significant at p = 0.0080 calculated using a two-tailed student's test. The experiment was repeated three times with consistent results.

Representative immunofluorescent images of mH2A and HP1β protein expression in the nuclei of human fibroblasts at different replicative ages. LF1 cells were stained and images acquired as described in Experimental procedures. The mH2A antibody was directly conjugated to Alexa Fluor 647. HP1β was visualized using a secondary antibody conjugated to Cy3. Early passage: vigorously growing cultures within the first third of their replicative life span. Quiescent: early passage cells were contact inhibited and then cultured in 0.25% serum for 48 hr. Late passage: cultures within 8–12 population doublings of their terminal senescent arrest. Senescent: cultures whose cell numbers have not increased for a minimum of 2 weeks. Flow cytometric cell cycle profiles for early passage, quiescent and senescent cultures are shown in Fig. S2. Arrowheads indicate the variety of SAHF morphologies seen in LF1 cells: cell showing robust mH2A and DAPI foci, panels D,H; cell showing intermediate but poorly resolved HP1β and DAPI foci, panels K,O; cell showing very few and small HP1β foci and no DAPI foci, panels L,P; cell showing no mH2A or DAPI foci but high overall levels of mH2A, panels C,G.

Immunoblotting analysis of total cellular extracts. Left panel: representative images from one experiment. Lanes were loaded for equivalent cell numbers. Note the clear upregulation of the cyclin dependent kinase inhibitors p16 and p21 in late passage cells, indicative of entry into cellular senescence. Right panel: immunoblots were quantified using the LI-COR Odyssey infrared imaging system. mH2A and HP1β levels were normalized to H2B levels detected in the same lane and are expressed as a ratio of mH2A or HP1β to H2B a.u. The values shown are the means of three separate experiments. Error bars indicate the SEM. The differences in expression between early and late cells are significant at p = 0.0032 and p = 0.042 for mH2A and HP1β, respectively, calculated using a two-tailed student's test.

Association of mH2A with pericentromeric heterochromatin. Representative images of single nuclei are shown at high magnification for (A) mouse liver and (B) mouse lung. Panels show the same nucleus stained for mH2A (top row, A,B), centromeres (middle row, C,D) and DAPI (bottom row, E,F). A young nucleus is shown on the left (A,C,E), and an old nucleus on the right (B,D,F). Scale bars designate 2 μm. Note that lung nuclei are considerably smaller than liver nuclei and are shown at higher magnification. (C) Quantification of mH2A-staining pericentromeric foci. A cell was considered positive if it displayed at least one mH2A focus colocalizing with a DAPI focus. At least 200 nuclei were scored for each condition. Error bars indicate the SEM.

Quantitative single cell measurements of mH2A protein levels in baboon skeletal muscle. (A) Representative images of sections from 5 and 25–30 years old vastus lateralis muscle. Animals of both sexes were included. (B) Immunofluorescent images collected in the experiment shown in panel A were quantified as indicated in Experimental procedures, expressed as ratios of mH2A and DAPI intensities for each nucleus, and plotted as histograms of cell number (% of total) against fluorescence intensity in arbitrary units (a.u.). Representative histograms for 2 young (blue) and 2 old (red) animals are shown. Note the prominent biphasic profile of the histograms. (C) Quantification of mH2A levels detected by immunofluorescence. The number of cells (% of total) under the low (green) and high (yellow) intensity peaks were calculated for each histogram, and means were calculated for the young and old age groups. A total of 5 young and 6 old animals were included in the analysis. Error bars indicate the SEM. The ratio of low to high intensity cells was 1.45 for the young animals, and 0.44 for the old animals. The difference between the ratios of low and high intensity cells is significant at p = 0.0018 calculated using a two-tailed student's test. The experiment was repeated three times with consistent results.