Analysis of expression, heterochromatin proteins, DNA methylation, and histone modifications in satellites. (A and B) FISH to sat II RNA (green) shows no increased expression with SADS formation in senescent (left) versus cycling (right) cells, with satellite chromatin marked by CENP-B staining (red). (C) CENP-A (green) and CENP-B (red) are still present in senescent cells as determined by the presence of SAHF (DAPI) and decondensed CENP-B staining (magnified in inset). (D) Comparison of H3K9me3, H3K27me3, and H3K4me3 read densities between cycling and senescent IMR90 cells and between cycling IMR90 and H1hES cells on α-sat (RepeatMasker class: HSATII) sequences with corresponding Pearson’s r values. (E and F) As all cancer samples examined, U2OS cells have compact satellites (F; α-sat, green; sat II, red) and do not form SADS unless treated with 5-AzaC, which induces senescence (E). (G) Quantification of mean 1q12 signal areas (n > 60) in different cell samples (error bars represent standard error). (H) A DNA methylation-sensitive Southern blot with U2OS and cycling Tig1 cells digested by BstB1 (B), HpaII (H), or MspI (M) and detected with a probe for sat II 1q12 sequences. The arrow points to a band size that is lacking in some of the methylation-sensitive HpaII lanes, indicating that the 1q12 region is methylated in the corresponding cell types. (I–K) Cells with normal levels of LaminB1 (red; top left) rarely contain distended α-sat (green; I and K), but in cells with SADS (bottom right) LaminB1 is often diminished (J and K; n = 100 from one of three replicates).