Fig. 2: H3.1 enrichment at chromocenters follows chromocenter replication status.

a Experimental scheme for visualizing endogenous H3.1/2 or H3.3 during the S phase. Aurora B and EdU staining allowed to resolve the cell cycle stage of individual cells. Cells are scored as G1 (negative to Aurora B and EdU), S phase (EdU positive), or G2 (negative to EdU and positive to Aurora B). S-phase designations are based on EdU S phase patterns with the Early S phase (E) defined by a diffused staining (euchromatin); Mid S phase (M) by a focused EdU labeling around DAPI foci (chromocenters); and late S phase (L) by specific foci staining at the nuclear periphery. b Left: Representative immunofluorescence images of ESCs after in vivo labeling with EdU (green), immunofluorescence staining of H3.1/2 and H3.3 (red), Aurora B (magenta), and DNA (DAPI, cyan). Scale bars, 10 μm. c Quantitative analysis of the proportion of cells displaying H3 variant enrichment patterns at PHC in different cell lines (ESCs, NPCs, NIH-3T3) during the G1, S, and G2 phases. Stacked histogram show the mean (in %) and bar standard deviation from 4 experiments for H3.1 in ESCs and 3 experiments for the other. For H3.1, n = 523, 329 and 275 nuclei for ESCs, NPCs, and 3T3, respectively. For H3.3 n = 328, 298, and 266 nuclei for ESCs, NPCs and 3T3 respectively. ANOVA two way test was used for statistical analysis: ns (p > 0.05), *(p < 0.05), **(p < 0.01). P values are shown in the source data file. Source data are provided as a Source Data file. d Schematic of FUCCI ESCs allowing visualization by microscopy of hCdt1-mCherry and Geminin-Citrine during the cell cycle. Cells are scored as G1 (mCherry++/Citrine−), Late G1/Early S (mCherry+/Citrine+), Mid/Late S (mCherry−/Citrine+), or G2 (mCherry−/Citrine++). e Representative immunofluoresce images of FUCCI ESCs expressing hCdt1 (red) or Geminin (green) with immunofluorescent staining of H3.1/2 (magenta) in G1, Early S, Late S, and G2 phases. Scale bars, 10 μm. f Quantitative analysis of the percentage of FUCCI ESCs exhibiting recurrent H3.1/2 patterns at chromocenters throughout the cell cycle. Bar plots show the mean and standard deviation of 100 nuclei for each cell cycle phase from 4 experiments. Source data are provided as a Source Data file.