Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments. (C) Progressive loss of core histones in senescent IMR90 cells in replicative senescence (RS). Lysates were normalized by cell number and an equal number of cells was loaded per each lane. Lamin A/C was used as a confirmatory loading control. See Materials and methods for details of time course. (D) Immunohistochemistry for histone H3 in human dermal nevus (n) and adjacent epidermis (e). Right-hand panels are expanded from boxed areas on the left. Note decreased nevus staining for H3 (right bottom) as compared with epidermis (right top). Bars: (left) 100 µm; (right) 50 µm. (E) Immunofluorescence of histone H3 (red) and S100 (melanocytes [green]) in human benign nevus and adjacent epidermis. Bars, 20 µm. (F) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus (bottom panels), compared with epidermal melanocytes (top panels). Bars, 20 µm. (G) Quantitative immunofluorescence of H3 in epidermal and nevus melanocytes. Images were obtained in blue (DAPI), green (S100), and red (H3) channels and then H3 intensity was measured in either S100⁺ epidermal cells, strictly adjacent to the basal membrane, or in S100⁺ nevus melanocytes. H3 intensity histograms represent fluorescence intensity distribution, combined from three individual nevi. At least 150 epidermal (50 per nevus) and 300 nevus (100 per nevus) melanocytes were assessed.