Fig. 2

Glutamylation is required for the maintenance of mouse and human pluripotency. a Mouse ESC D3 cells were transfected with scrambled shRNA (shCtrl), shCCP1, shCCP6 or CCP1 and CCP6 overexpression (oe) plasmids as indicated and cultured in mouse ESC media. After 5 days, pluripotency was analyzed by AP staining. Colony numbers for undifferentiated, mixed or differentiated clones were calculated as means ± S.D. **P < 0.01. Scale bar, 100 μm. n = 4. b D3 cells were transfected with the indicated plasmids and assessed AP staining as in a. **P < 0.01. Scale bar, 50 μm.n = 6. c Embryos at the indicated stages were isolated and stained with GT335 antibody and PI, and visualized by confocal microscopy. Scale bar, 20 μm. Percentages of cells with GT335 signal localized in the nucleus were counted as means ± S.D. **P < 0.01. For 1-cell-stage embryos, n = 48. For 2-cell-stage embryos, n = 56. For 4- to 8-cell stage embryos, n = 62. d Human ESC H9 cells were infected with lentivirus expressing the indicated shRNAs and cultured in human ESC media for 3 weeks. Pluripotency was analyzed by AP staining. Scale bar, 100 μm. AP-positive colony numbers per well were calculated as means ± S.D. **P < 0.01. n = 5. e Depletion of CCP1 or CCP6 in human iPSCs increases AP⁺ colony formation. Human iPSCs were infected with lentivirus expressing the indicated shRNAs and cultured in hiPSC media for 3 weeks, followed by AP staining. Scale bar, 50 μm. AP-positive colony numbers per well were calculated as means ± S.D. **P < 0.01. n = 5. f CCP1 or CCP6 depletion in human iPSCs promotes teratomas formation. Human iPSCs infected with the indicated lentivirus were injected subcutaneously into NOD/SCID mice. Six weeks later, teratomas were collected for H&E staining. Red arrow denotes teratomas in upper panels. Black arrow, endoderm; red arrow, mesoderm; red arrowhead, ectoderm. Scale bar, 200 μm. Total 6 teratomas from 6 mice were analyzed pre condition (n = 6). The teratomas were from 2 iPSC cell lines. Student’s t test was used as statistical analysis. ns no significance