Fig. 6

Tbx3 meditates the self-renewal-promoting effect of Rhox6 knockdown in mESCs. A qRT-PCR analysis of the expression of Rhox6 and Tbx3 in scramble and Rhox6 shRNA mESCs infected with scramble or Tbx3 shRNA lentiviruses. The data are presented as the mean ± SD (N = 3 biological replicates). *P < 0.05, **P < 0.01 versus scramble, ^##P < 0.01 versus Rhox6 sh#1/scramble, as determined by one-way ANOVA with Sidak’s multiple comparisons test. B Western blot analysis of the protein levels of Tbx3 in scramble and Rhox6 shRNA mESCs infected with scramble or Tbx3 shRNA lentiviruses. C AP staining and immunofluorescence staining of Klf4 in scramble and Rhox6 sh#1 mESCs infected with scramble or Tbx3 shRNA lentiviruses and cultured in serum-containing medium without LIF for 7 days. Scale bar, 100 μM. D qRT-PCR analysis of the expression of pluripotency- and differentiation-associated genes. The data are presented as the mean ± SD (N = 3 biological replicates). *P < 0.05, **P < 0.01 versus scramble, ^#P<0.05,  ^##P<0.01 versus Rhox6 sh#1/scramble,  as determined by one-way ANOVA with Sidak’s multiple comparisons test. E CUT&Tag analysis of the binding sites of Rhox6 on the Tbx3 promoter. F Ten pairs of qRT-PCR primers were designed using the Tbx3 promoter as the template. G A ChIP assay was performed using a Flag antibody. IgG was used as a negative control. The fold enrichment in the indicated regions of the Tbx3 promoter was measured by qRT-PCR. The data are presented as the mean ± SD (N = 3 biological replicates). **P < 0.01 versus IgG, as determined by Student’s t test. H Binding position and sequence of Rhox6 on the Tbx3 promoter and the corresponding deletion mutation sequence. I Luciferase activity analysis of the WT or mutant (Mut) Tbx3 promoter reporter plasmid-expressing cell lines transfected with or without PB-Rhox6. PB/Tbx3^WT was used as the control for normalization. The data are presented as the mean ± SD (N = 3 biological replicates). *P < 0.05, **P < 0.01 versus PB/Tbx3^WT, ^##P < 0.01 versus PB-Rhox6/Tbx3^WT, as determined by one-way ANOVA with Sidak’s multiple comparisons test