Figure 4: Usp16^−/− ESCs are defective for differentiation.

(a) Phase-contrast images of EB morphology during EB formation from Usp16^+/+ and Usp16^−/− ESCs. (b) Growth curve of EBs formed from Usp16^+/+ and Usp16^−/− ESCs. The diameter of EBs at each day was measured and compared. For each time point, 50 EB diameters were measured. Means and s.d. (error bars) are shown. (c) Haematoxylin and eosin staining of day12 EBs formed from Usp16^+/+ and Usp16^−/− ESCs. Day 12 EBs from Usp16^−/− ESCs lack organized structure and failed to differentiate. Scale bar, 100 μm. (d) Western blot analysis of the levels of Usp16 (top panel), ubH2A (sixth panel) and pluripotent genes (second to fourth panels) during EB formation. ubH2A signals were quantified as in Fig. 2c. Signals in Usp16^+/+ EBs at day 0 were arbitrarily set as 1. GAPDH and histone H3 were used as loading controls. Original scans for blots are shown in Supplementary Fig. 7. (e) Real-time PCR analysis of Usp16 and pluripotent genes expression at day 0, day 6 and day 12 during EB formation of two independent Usp16^+/+ and Usp16^−/− ESC lines. Gene expression levels at day 0 were arbitrarily set as 1. Bars represent means+s.d. Number of biological replicates n=3. (f) Real-time PCR analysis of lineage-specific genes expression at day 0, day 6 and day 12 during EB formation from two independent Usp16^+/+ and Usp16^−/− ESC lines. Gene expression levels at day 0 were arbitrarily set as 1. Bars represent means+s.d. Number of biological replicates n=3.