Fig. 2.

Human iPS cells lacking MBD3/NuRD fail to undergo programmed differentiation. A) Western blot of nuclear extracts from wild type (WT), MBD3-KO and MBD3-KO hiPSCs rescued with an MBD3-3xFLAG transgene (“Rescue”). The blot was probed with antibodies indicated at left. The closed arrowhead indicates native MBD3, while the open arrowhead indicates the MBD3-3xFLAG fusion protein. B) Nuclear extracts from wild type, MBD3-KO and Rescued cells was immunoprecipitated with anti-Chd4, western blotted and probed with antibodies indicated at right. Arrowheads as in Panel A. C) Scheme of the differentiation experiment (top) and images of indicated cell cultures at Day 0, 7 or 20 of differentiation. Scale bar indicates 100 µm. D) Expression of pluripotency (POU5F1, SOX2 and NANOG) and lineage specific genes (FOXG1, PAX6 and NESTIN) during neural differentiation was measured by qRT-PCR. Y-axis represents expression relative to that in wild type cells at Day 0, while the X-axis represents the time in days. Error bars represent the standard deviation of ≥3 biological replicates. E) as in panel D, but for definitive endoderm differentiation protocol. Pluripotency-associated genes (POU5F1, NANOG, ZFP42) on the left, and differentiation-associated genes (GATA4, FOXA2, SOX17) at right.