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. 2015 Oct 1;8(10):1295–1309. doi: 10.1242/dmm.020594

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — OTX2 overexpression significantly decreases levels of hESC genes. (A) Results from global gene expression analysis demonstrating overall downregulation of hESC pluripotency genes in OTX2-overexpressing hENs. Graph represents n=3 biological replicate pairs of OTX2 hEN and hENs. All transcripts are significantly different. (B) Upregulation of representative miRNA targets of LIN28A following OTX2 overexpression in hENs. n=3 independent biological replicates. (C,D) Validation of hESC transcript downregulation in hENs (C) and Daoy (D) following OTX2 overexpression by qPCR. Error bars: s.e.m. *P<0.05, **P<0.01, ***P<0.001. (E-H) Comparative ChIP-qPCR analysis for OTX2 binding and histone modifications (H3K4me3 and H3K27me3) on the OCT4, NANOG, LIN28A and SOX2 promoters in OTX2+ Daoy versus Daoy cells. Error bars: s.d. (I,J) Luciferase activity in OTX2+ Daoy (I) and OTX2+ hEN (J) following transfection of a SOX2 promoter reporter construct. Error bars: s.e.m. Luciferase activity was normalized relative to control Daoy or hEN. (K,L) Western blots depicting decreased levels of SOX2 in Daoy SHH MB (K) and hEN cells (L) following OTX2 overexpression. β-actin serves as a loading control.