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. 2019 Nov 13;47(22):11637–11648. doi: 10.1093/nar/gkz1040

Figure 1.

Figure 1.

LmaH3 is incorporated into HeLa cell chromatin. (A) Amino acid sequence alignment of histone H3s from Leishmania major [LmjF.10.0870 (XP_001681421.1)] and Homo sapiens (NP_003520.1). The black boxes indicate identical amino acid residues between H3.1 and LmaH3. (B) Schematic representation of the fractionation of micrococcal nuclease digested chromatin. (C–E) Sucrose gradient ultracentrifugation. The chromatin fractions from HeLa cells stably expressing LmaH3-GFP (C), H3.1-GFP (D), or untransfected cells (E) were subjected to sucrose gradient ultracentrifugation. The resulting DNA fragments were analyzed by agarose gel electrophoresis with ethidium bromide staining. The DNA markers are indicated as M. (F) Detection of LmaH3-GFP by western blotting analysis. The presence of H3.1-GFP and LmaH3-GFP was detected by a western blotting analysis, using the anti-GFP monoclonal antibody. Samples (10 μl each) of HeLa cells expressing H3.1-GFP (fraction 10), untransfected cells (fraction 10), and HeLa cells expressing LmaH3-GFP (fraction 10), were applied (upper panel). To detect the low amount of LmaH3 incorporated into chromatin, samples (fraction 10) from LmaH3-GFP cells and untransfected cells (as a negative control) were concentrated 13-fold, and then subjected to the western blotting. The sample from HeLa cells expressing H3.1-GFP (fraction 10) was not concentrated. The results were reproduced, and are represented in Supplementary Figure S1. The molecular weights of the marker proteins are indicated. The full gel images of Figure 1C–F are presented in Supplementary Figure S5A–E, respectively. (G and H) Fluorescence recovery after photobleaching (FRAP) analysis of HeLa cells expressing H3.1-GFP and LmaH3-GFP. After bleaching a rectangular area of the nucleus, the mobility of H3.1-GFP and LmaH3-GFP in living cells was analysed by monitoring the fluorescence recovery. (G) Representative images before photobleaching (left column), upon bleaching at 0 min (centre column), and at 2.5 min (right column) are shown. The images for HeLa cells expressing H3.1-GFP and LmaH3-GFP with fast (rapid LmaH3-GFP) or slow (slow LmaH3-GFP) fluorescence recovery are presented in the upper, middle, and lower panels, respectively. The scale bar indicates 4 μm. (H) Graphical representation of the FRAP data. The relative fluorescence intensities of H3.1-GFP (•), rapid LmaH3-GFP (◊), and slow LmaH3-GFP (○) are presented with their standard deviations (n = 10).