Fig. 7

Deletion of TTLL1 or TTLL4 impairs iPSC induction and ESC pluripotency. a Depletion of TTLL1 or TTLL4 impairs iPSC formation. TTLL1 or TTLL4 was silenced in 4F2A MEFs as well as rescued with TTLL1 or TTLL4 into their silenced MEFs, followed by dox treatment for iPSC formation and stained with anti-Nanog antibody as in Fig. 1e. Scale bar, 50 μm. Nanog-positive colony numbers per 10⁴ cells were calculated and shown as means ± S.D. **P < 0.01. n = 5. b Immunoblotting of Klf4 in above-treated MEFs as in a. Fold changes of relative expression of indicated proteins compared with β-actin were caculated as means ± S.D. The data represent four independent experiments. c TTLL1 or TTLL4 depletion enhances the pluripotency of mouse ESCs. D3 cells were transfected with scrambled shRNA (shCtrl), shTTLL1, shTTLL4, or TTLL1 and TTLL4 overexpression (oe) plasmids 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. n = 5. Scale bar, 100 μm. d D3 cells were transfected with indicated plasmids. mRNA levels of the indicated genes were analyzed by real-time qPCR. Relative gene expression fold changes were counted as means ± S.D. **P < 0.01. n = 5. Primer pairs are shown in Supplementary Table 1. e Indicated plasmids were transfected into mouse D3 cells as in c. Colony numbers for undifferentiated, mixed, or differentiated clones were calculated as means ± S.D. n = 5. Scale bar, 100 μm. f mRNAs levels of the indicated genes were analyzed by real-time qPCR as in d. Relative gene expression fold changes were counted as means ± S.D. **P < 0.01. n = 5. g TTLL1 or TTLL4 depletion in human ESC H9 cells was confirmed by real-time qPCR. n = 5. h TTLL1 or TTLL4 depletion in human ESC H9 cells increases AP⁺ colony formation. Human H9 cells were infected with lentivirus expressing the indicated shRNAs and cultured in human ES media for 3 weeks. ESC pluripotency was analyzed by AP staining. AP⁺ colony numbers were calculated as means ± S.D. n = 4. Scale bar, 100 μm. Student’s t test was used as statistical analysis. oe overexpression