Figure 11

H4-K16Ac and HMGA proteins act synergistically in DNA compaction leading to the formation of striking senescence-associated heterochromatin foci (SAHFs). A) Deconvoluted mass spectra of intact histone H4 in proliferating and RAF-senescent RPEhTERT. B) DNA 4',6'-diamidino-2-phenylindole (DAPI) staining of representative nuclei and corresponding DAPI coefficient of variation (CV) values. C) Boxplots of DAPI CV (n > 80, from one experiment). *DNA compaction statistically different from retinal pigmented epithelial (RPE) Prolif. (P < 10^-5, Welch t-test). D) SDS-PAGE of acid-extracted proteins from proliferating and oncogene-induced senescent WI-38hTERT, IMR-90, BJ and RPEhTERT cells. E) Immunoblot of extracts from RPEhTERT/RAF-ER cells treated with siRNA (24 hours) and 20 nM 4-HT (6 days) using anti-MOF, -GAPDH (loading control), -H4-K16Ac, -H3 (loading control) antibodies. F) DNA DAPI staining of representative nuclei and corresponding DAPI CV values. G) Boxplots of DAPI CV (n > 50, from one experiment). *DNA compaction statistically different from No Target - pWZL Ctl (P < 10^-5, Welch t-test). WI-38 SenRAF: WI-38hTERT/GFP-RAF-ER + 4-HT 20 nM (5 days); IMR-90 SenRAS: IMR-90/ER-RASval12 + 4-HT 100 nM (6 days); BJ SenRAS: BJ/ER-RASval12 + 4-HT 100 nM (5 days); RPE SenRAF: RPEhTERT/GFP-RAF-ER + 4-HT 100 nM (6 days).