Fig. 2. Temporal proteome changes during entry into pausing in ESCs.
a, Immediate and adaptive protein expression changes in ESCs. k-Means clustering was performed on differentially expressed proteins identified with MetaboAnalyst. b–d, GO analysis of upregulated (b) and downregulated (c) proteins identified in a. Immediately upregulated pathways pertain to chromatin organization, whereas metabolic pathways show an adaptive response. GO analysis of proteome changes in TSCs (d). The full list of significantly enriched GO terms is provided in Supplementary Table 3. The Benjamini–Hochberg correction was used. P value cutoff 0.05 and q value cutoff 0.1. e, TEM images of selected areas with one or more nuclei in the epiblast and TE of E4.5 and diapaused embryos. The nucleus is denoted with ‘N’ and visible as the area with an electron dense periphery, while the nucleolus is denoted with ‘n’. Scale bar, 2 µm. f,g, IF staining of H3K9me2 and Lamin B1 in E4.5 and diapaused (EDG7.5) embryos. Projections at 20× (f; scale bar, 50 µm), and close-ups at 63× (g; scale bar, 5 µm), are shown. The ICM is indicated by the dashed line. h,i, Quantifications (h) of the H3K9me2 and Lamin B1 signal intensity along single-cell cross-sections (i), normalized for DAPI (n = 5–8 cells). ICM and TE cells of E4.5 and EDG7.5 embryos were quantified along the lines shown in i (scale bar, 5 µm). The signal intensity of H3K9me2, Lamin B1 and DAPI were quantified per cell, using the multichannel plot profile of the BAR plugin in Fiji-2. The cell size was scaled, and DAPI-normalized H3K9me2 and Lamin B1 intensity were plotted. Data are presented as median with confidence interval.