Figure 8One thousand ESCs of the indicated genotypes were placed into hanging-drop cultures to form EBs. After 3 days (referred to as day-0), differentiation was induced by plating EBs onto gelatin coated tissue culture dishes in differentiation-inducing medium. (A) Principal Component Analysis (PCA) of the proteome profiles of Tnfaip2-/- (KO) versus WT ESCs and EBs on day-0 to day-3 after differentiation induction (n=5 biological replicates per group, enlarged symbol denotes the mean). (B) The Volcano plots show the differentially expressed proteins identified from the proteome analysis of Tnfaip2-/- EBs versus WT EBs at the indicated days after induction of differentiation. Vim (marked in blue asterisk) was the only protein that featured among top 100 differentially expressed proteins at all time points by implementing selection criteria of an average log2ratio > +/-1.2 and a -log10Qvalue <2. Vim expression was reduced at all timepoints of in vitro differentiation in Tnfaip2-/- EBs vs. WT EBs (n=5 biological replicates, see also Dataset EV8 for top 100 regulated proteins). Sphere size illustrates the magnitude of log2fold changes as does color coding: blue-downregulated proteins, red-upregulated proteins; log2fold marking by color intensity: strong intensity >2, medium intensity > 1.5, faded color >1. (C) mRNA expression of Vim on indicated days of WT EB and Tnfaip2-/- EB differentiation (n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple testing). FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (D) Representative FACS blot of BODIPY staining. Unstained WT EBs served as a negative control (grey). FACS analysis of lipid droplet (LD) content of WT (blue) and Tnfaip2-/- (red) EBs by BODIPY493/503 staining at the indicated days after differentiation induction: (E) Quantification of staining intensity [n=3 independent cultures per genotype; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by multiple t-tests with Holm-Sidak correction for multiple comparisons]. Transmission Electron Microscopy (TEM) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2-/- ES cells on day-1 after differentiation induction. (n=5 independent cultures per genotype, n=100 images per replicate). (F) Representative micrographs of TEM analysis of EB cells of the indicated genotype. Yellow arrows point to LDs, scale bars: 1 µm. Electron Microscopy (TEM) was used to determine the number of LDs in EB cultures derived from WT and Tnfaip2-/- ES cells on day-1 after differentiation induction. (n=5 independent cultures per genotype, n=100 images per replicate). (G) Quantification of LDs per TEM field. Data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney test. (H) UPLC-MS/MS analysis revealed a reduction in triacylglycerol (TAG) content in Tnfaip2-/- EBs compared to WT EBs at the indicated time points after differentiation induction [n=3 independent cultures of EBs per time point; data of EBs on day-0 to day-3 of differentiation were statistically analyzed; mean centered group data were normally distributed and (P>0.05 as per Shapiro-Wilk test) and analyzed by one-sided t-test with Holm Sidak correction for multiple testing].