Fig. 4

The deubiquitin activity of USP8 is required for ESC identity maintenance. a,b Western blot analysis of whole-cell extracts from MEFs and ESCs using anti-USP8 antibody. β-Actin served as a loading control. Images are representative of three independent experiments. Error bars indicate the SD (n = 3). Three independent biological replicates. *P < 0.05 by unpaired two-tailed Student’s t test. c, d Colony-formation assay of Usp8^+/+ and Usp8^▵/− ESCs. ESC colonies were stained by AP. Error bars indicate the SD (n = 3). Three independent biological replicates. **P < 0.01 by unpaired two-tailed Student’s t test. e ESC pluripotency is impaired by decrease of Usp8. The relative mRNA expression of pluripotency genes in Usp8^+/+ and Usp8^▵/− ESCs was assayed by quantitative PCR. Error bars indicate the SD (n = 3). Three independent biological replicates. *P < 0.05; **P < 0.01 by unpaired two-tailed Student’s t test. f Schematic illustration of the ___domain organization of USP8 and the ___location of the C748A mutation. Boundaries of individual domains and the amino acid numbering of the catalytic Cys residue are indicated according to mouse USP8. USP, ubiquitin-specific protease ___domain with catalytic Cys 748. MIT-Rhod (amino acid residues 1–319). g,h The reduced colony formation ability of Usp8^▵/− ESCs is rescued by introduction of WT Usp8, but not the C748A Usp8 mutant. Error bars indicate the SD (n = 3). Three independent biological replicates. NS not significant. *P < 0.05 by unpaired two-tailed Student’s t test. i Introduction of WT but not the C748A Usp8 mutant into Usp8^▵/− cells restores the expression of pluripotency genes. The relative mRNA expression of pluripotency genes was determined by quantitative PCR. Error bars indicate the SD (n = 3). Three independent biological replicates. NS not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ^#P < 0.05, ^##P < 0.01 by unpaired two-tailed Student’s t test. # (Usp8^∆/- +∆C748A compared with Usp8^+/+)