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Fig. 3 Nanog transcriptionally inhibits the expression of eef1a1l2. (A) RNA-seq analysis showing significantly increased expression of eef1a1l2 in nanog null eggs. Red dots indicate upregulated genes, green dots indicate downregulated genes, and gray dots indicate genes that were not differentially expressed in nanog mutant eggs. (B) Detection of eef1a1l2 expression in nanog mutant oocytes at different stages revealed by RT-qPCR. ***P<0.001. n=3. (C) Detection of eef1a1l2 expression in ovaries of WT, nanog mutant, and Tg(CMV:nanog-myc) in nanog mutant background by in situ hybridization on cryosections. Scale bar: 200 μm. (D,E) WISH (D) and RT-qPCR (E) analysis showed reduced expression of eef1a1l2 in nanog or En-nanog overexpressed embryos at 6 hpf. Images in D are representative of 36/36 embryos for WT, 38/39 for +EN-nanog and 32/32 for +nanog. **P<0.01. n=3. Scale bar: 100 μm. (F) ChIP analysis of Tg(CMV:nanog-myc) ovaries with anti-Myc antibody at 6 months post-fertilization. The promoter region of eef1a1l2 was enriched in precipitated chromatin. rpl5b served as a negative control. Schematic depicts the genomic sequence of eef1a1l2, and the sequence located between −3748 and −3496 bp upstream of the amplified region.