Fig. 3: Widespread proteome alteration is largely associated with transcriptional rewiring.

a Principal component analysis of the RNA-seq and proteome data from ESCs collected at different time points of the 2iL to SL and EPI transition. n = 2 biological replicates per time point for RNA-seq and n = 3 biological replicates per time point for total proteome, each dot represents one sample. b Volcano plots showing the label-free total protein quantification in 2iL, SL, and EPI conditions. Values represent the mean of three biological replicates. Representative examples are shown. Differentially expressed proteins (FDR < 0.05 and FC ≥ 3) are highlighted in black. Differential expression was calculated using the two-sided t-test. c Venn diagram showing the overlap of differentially expressed proteins in SL and EPI when compared to 2iL ESCs. N numbers represent proteins and are stated in the figure. d Graphs showing the percentage of differentially expressed proteins with or without change in RNA/RFP when SL or EPI are compared to 2iL state. Transcriptional: protein change >3-fold and RNA change >2-fold; translational: RFP change >2-fold and RNA change <2-fold; post-translational: RNA and RFP change <2-fold. e Heat-map showing the change in RFPs, RNA, and protein in different categories defined in panel c. n = 194 differentially expressed proteins. Values represent mean of log2 fold change. f Box plots showing the changes in RNA, RFP, and protein levels for the post-translationally regulated genes identified in panel c and during the 2iL-to-SL and EPI transition. n represents the number of proteins as indicated in the figure. Box=25–75th percentile; bar=median; whiskers=5–95th percentile. g Expression pattern of differentially expressed proteins in at least one time point of 2iL to Sl and EPI transition. Lines in the plots represent average fold-change of protein expression and shaded bands represent upper 75% and lower 25% quantiles.