Figure 2: Usp16 knockout does not affect ESC viability and identity.

(a) Schematic representation of the strategy used to delete Usp16 (top panels) and an image of genotyping result of Usp16 deleted ESCs (bottom panel). PCR primers used for genotyping are indicated in the top panel. (b) Phase-contrast images (top panels) and alkaline phosphatase staining images (bottom panels) of Usp16^+/+ and Usp16^−/− ESCs. Scale bar, 50 μm. (c) Western blot analysis of Usp16 (top panel), H2A ubiquitination (third panel) and H2B ubiquitination (fifth panel) levels in two independent Usp16^+/+ and Usp16^−/− ESC lines. The quantification of ubH2A signals was labelled. Signals in control Usp16^+/+ ESCs were arbitrarily set as 1. GAPDH, histone H2A, H2B and H3 were used as loading controls. (d) Western blot analysis of the levels of pluripotent genes and PRC subunits in two independent control Usp16^+/+ and Usp16^−/− ESC lines. β-tubulin was used as a loading control. (e) Growth curve of two independent control Usp16^+/+ and Usp16^−/− ESC lines. A total of 1 × 10⁴ ESCs were seeded in 12-well plates and cell numbers were counted every other day. The same seeding procedure was repeated every other day. (f) Fluorescence-activated cell sorting (FACS) analysis of control Usp16^+/+ and Usp16^−/− ESC lines. The percentages of cell populations at each cell cycle phase were labelled. Original scans for gels and blots are shown in Supplementary Fig. 7.