Figure 2. Heterochromatin levels decline with aging.

(A) Gut tissues from young (3-day old) and old (35-day old) female flies were dissected and stained with anti-HP1 (magenta). Images were scanned at identical settings with confocal microscopy. Note that HP1 forms prominent foci in the young gut (one pointed by an arrow), whereas in the old gut HP1 staining seems more diffuse and lacks the prominent foci. (B) Male flies of indicated age (in weeks) were homogenized and the protein extracts were subjected to SDS-PAGE and blotted sequentially with antibodies for H3K9m2, HP1, H3, and α-Tubulin. Representative images for one of the three experiments are shown. Lower panels show intensity ratios as indicated. Note that total H3 levels decrease with age, and that H3K9m2 signals decrease with age even when normalized to total H3. (C) Chromatin immunoprecipitation (ChIP) was carried out with anti-HP1 antibodies using extracts from young (3-day old) and old (35-day old) male wild-type or hsp70-HP1/+ flies. Note that HP1 is enriched in 1360 (representative heterochromatin sequence) of young but not old wild-type flies (lane 2, top two panels), and is detectable in both young and old hsp70-HP1/+ flies (middle panels). (D) Ovaries from young (3-day old) and old (35-day old) female DX1 flies were stained with anti-ßgal. Note the much increased ßgal levels in old ovaries. (E) Total protein from single young (3-day old) and old (35-day old) female flies of indicated genotypes were blotted by anti-ß-gal antibodies or anti-aTublin (control). Note the appearance of ß-gal in old DX1 flies (arrow). The band below ß-gal is a nonspecific band.