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. 2010 Jan 11;285(11):8408–8421. doi: 10.1074/jbc.M109.064386

FIGURE 3.

FIGURE 3.

Analysis of the Oct25ΔPOU(273–301) effect on gene transcription. A, injection of 1 ng of Oct25ΔPOU(273–301) RNA leads to an up-regulation of genes that induce mesoderm and endoderm and dorsalize body axis but does not generate an appreciable effect on the genes in the BMP pathway (BMP4, Xvent1, and Xvent2). B, injection of 1 ng of Oct25ΔPOU(273–301) RNA also increases the transcription of mesendoderm inducing genes and germ layer dorsalizing genes in animal caps. C, Oct25ΔPOU(273–301) stimulates luciferase reporter activity for the promoters of Gsc (GscLuc(−1500)), Xnr1 (Xnr1Luc(−907)), Xnr3 (Xnr3Luc), Sia (SiaLuc(−802)), the artificial promoter composed of six repeats of the distal element on Gsc promoter (6×DE), and the Wnt-responsive artificial promoter reporter TopFlash. For control, Oct25ΔPOU(273–301) does not have any strong effect on the pGL3-basic vector or on FopFlash, the negative control reporter for TopFlash. In each luciferase assay, 40 pg of reporter plasmid and 400 pg of RNA were injected. D–F, induction of genes by Oct25ΔPOU(273–301) is severely compromised after blocking the activities of VegT, activin/nodal, and Wnt/β-catenin signaling pathways. D, specific knockdown of VegT by 40 ng of an antisense morpholino (VegTMO) leads to reduced transcription of Gsc, Chd, Xnr1, Xnr2, and Sia. Injection of 1 ng of Oct25ΔPOU(273–301) RNA results in strong up-regulation of these genes. Up-regulation is lost when the RNA is co-injected with VegTMO. E, blocking of the nodal/activin signaling pathway by injection of 800 pg of a dominant-negative activin receptor I (dnXAR1) reduces the transcription of genes, like Gsc, Chd, Xnr1, and Xnr2, although injection of 1 ng of Oct25ΔPOU(273–301) RNA stimulates these genes. This stimulation is weakened by co-injection of dnXAR1 and Oct25ΔPOU(273–301) RNAs. F, when the Wnt/β-catenin signaling pathway is inhibited by injection of 400 pg of dominant-negative TCF3 (dnTCF3) RNA, transcription of Chd, Gsc, Xnrs, and Sia is dramatically decreased. Consequently, co-injection of 1 ng of Oct25ΔPOU(273–301) cannot stimulate the transcription of these genes anymore.