Figure 6
From: Deacetylation of H4-K16Ac and heterochromatin assembly in senescence
SIRT2 contributes to deacetylation of H4-K16Ac and DNA compaction during RAF-induced senescence. A) Immunoblot of extracts from WI-38hTERT/GFP-RAF-ER cells treated with siRNA (24 hours) and 20 nM 4-HT (3 days) using anti-SIRT1, -SIRT2, -MOF, -GAPDH (loading control), -H4-K16Ac, -H3 (loading control) antibodies. Two independent experiments are shown for the depletion of SIRT2. B) Boxplots of 4',6'-diamidino-2-phenylindole coefficient of variation (DAPI CV) (n > 70, from one experiment). *DNA compaction statistically different from No Target (P < 10-5, Welch t-test). Biological replicates are shown Figure 7B. C) mRNA level of SIRT2 by qPCR after siRNA treatment (24 hours) and 20 nM 4-HT (6 hours). D) Immunoblot of WI-38hTERT/GFP-RAF-ER cells treated with 20 nM 4-HT for the indicated times using anti-SIRT1, -SIRT2, -MOF and -GAPDH (loading control) antibodies. Histogram shows the normalized level of SIRT2 to GAPDH for two independent time courses. E) Relative abundance of H4 acetylation states measured at the protein level on deconvoluted mass spectra. Error bars show SD of three biological replicates. F) Boxplots of DAPI CV (n > 60, from one experiment). *DNA compaction statistically different from Prolif. (P < 10-5, Welch t- test). Prolif.: proliferating WI-38hTERT/GFP-RAF-ER.