Fig. 4

Decreased Rhox6 levels promote mESC self-renewal. A qRT-PCR analysis of the expression level of Rhox6 in 46C mESCs infected with scramble and Rhox6 shRNA lentiviruses. The data are presented as the mean ± SD (N = 3 biological replicates). **P < 0.01 versus scramble, as determined by one-way ANOVA with Sidak’s multiple comparisons test. B Western blot analysis of the expression levels of Klf4 and Sox2 in 46C mESCs infected with scramble and Rhox6 shRNA lentiviruses and cultured in the absence of LIF for 7 days. C AP staining of scramble and Rhox6 shRNA-expressing mESCs in the absence of LIF for 7 days. Scale bar, 100 μM. D qRT-PCR was used to detect the expression of pluripotency and differentiation-related genes in scramble and Rhox6 shRNA mESCs. The data are presented as the mean ± SD (N = 3 biological replicates). *P < 0.05, **P < 0.01 versus scramble, as determined by one-way ANOVA with Sidak’s multiple comparisons test. E Immunofluorescence staining of Klf4 expression in scramble and Rhox6 shRNA-expressing mESCs. Scale bar, 100 μM. F qRT-PCR analysis of the expression of the Oct4, Rhox6, Elf5, Sox17, Cdx2, Mixl1, Gata4 and T genes in mESCs and EBs on different days. The data are presented as the mean ± SD (N = 3 biological replicates). *P < 0.05, **P < 0.01 versus D0, as determined by one-way ANOVA with Sidak’s multiple comparisons test. D0, Day 0