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A
Doubling time in pRH6 versus 2a2iL‐RH6. **P < 0.01 (t‐test). Data presented as mean ± SD (n = 3).
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B
Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL‐RH6. ROCKi (−) *P < 0.05, ROCKi (+) *P < 0.01 (t‐test), data presented as mean ± SD (n = 3).
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C
Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL‐RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co‐transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL‐RH6. OCT4 PE‐Luc ***P < 0.001, OCT4 DE‐Luc *P < 0.05 (t‐test). Data presented as mean ± SD (n = 3).
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D
qRT–PCR analysis of pluripotency‐related genes in pRH6 and 2a2iL‐RH6. *P < 0.05, **P < 0.01, ***P < 0.001 (t‐test). Data presented as mean ± SD (n = 3).
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E
Immunofluorescence staining for several naïve‐specific markers in 2a2iL‐RH6. Scale bar: 100 μm.
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F
Western blot analysis of naïve‐related pluripotency markers in pRH6 and 2a2iL‐RH6. **P < 0.01, ***P < 0.001 (t‐test). Data presented as mean ± SD (n = 3).
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G
Immunofluorescence staining against pRH5 and 2a2iL‐RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6‐diamidino‐2-phenylindole (DAPI). Reactivation of inactive X‐chromosome in female 2a2iL‐RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 μm.
