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. 2017 May 10;8:14995. doi: 10.1038/ncomms14995

Figure 1. H2A.J accumulates in senescent human fibroblasts with persistent DNA damage.

Figure 1

(a) Experimental plan. (b–f) WI-38hTERT-GFP-RAF-ER fibroblasts21 were induced into senescence by a hyperactive RAF kinase for 5 days (e-SenRAF) or 20 days (d-SenRAF), into quiescence by serum starvation for 5 days (e-Quiescent) or 20 days (d-Quiescent), or into senescence by treatment with 20 μM etoposide for 5 days (e-SenETO) or 20 days (d-SenETO). Histones and RNAs were extracted from these cells and from non-immortalized proliferating WI-38 fibroblasts (Early Passage) and the same cells maintained in replicative senescence for 20 days (d-SenRep). (b) Top-down deconvoluted mass spectra of intact histone H2A2 proteins showing accumulation of H2A.J (orange) only in samples derived from senescent cells with persistent DNA damage (d-SenETO and d-SenRep-see e below). Canonical H2A2 species are in grey (Supplementary Figs 1,2 for details). (c) Relative abundance of H2A.J versus all canonical H2A species determined by quantifying the Gly5-Arg12 tryptic peptide from H2A.J versus the same peptide from all canonical H2A species (Fig. 1d and Supplementary Fig. 2). Shown are the mean and s.d. for 4 biological replicates, except ‘Early Passage' and ‘d-SenRep' (2 biological replicates). (d) Sequence alignment of human H2A.J, H2A fraction 1 (H2A1), H2A fraction 2 (H2A2), and H2A.X. H2A.J is distinguished by Val11 and, like the H2A.X variant, by the presence of an SQ motif near the C-terminus (boxed in red). (e) Percent cells expressing the indicated number of γH2AX foci, a marker of DNA damage. Most SenETO and SenRep fibroblasts contained multiple foci, whereas most fibroblasts in proliferation, quiescence, or senescence induced by a hyperactive RAF kinase (SenRAF) contained no foci. Mean and s.d. from a minimum of 2 biological replicates. (f) Western blot of acid-extracted histones from fibroblasts as indicated. The H2A.J/H3 levels were normalized to the ratio in proliferation. (g) Time course of H2A.J accumulation during etoposide-induced senescence of WI-38hTERT fibroblasts by Western blotting. The error bars show the s.d. from three biological replicates.