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. 2012 May 4;10(5):583–594. doi: 10.1016/j.stem.2012.02.020

Figure 3.

Figure 3

Misexpression of NuRD Target Genes Contributes to the Differentiation Defect of Mbd3−/− ESCs

(A) Expression of indicated genes in wild-type and Mbd3−/− ESCs in serum and LIF or in the absence of LIF for the indicated times is plotted relative to expression in wild-type cells prior to LIF withdrawal. Error bars represent SEM from ≥3 experiments performed on different wild-type and mutant ESC lines. See also Figure S2.

(B) Expression levels of Klf4 (black bars) and Klf5 (white bars) in Mbd3 heterozygous ESCs (Flox/−) or Mbd3−/− ESC lines expressing microRNAs directed against Klf4, Klf5, and LacZ are displayed relative to expression levels seen in Mbd3Flox/− ESCs. Error bars represent SEM.

(C) An alkaline phosphatase (AP) assay was performed using Mbd3Flox/− ESCs (referred to as WT for simplicity), Mbd3−/− ESCs (KO), and two different Mbd3−/− ESC lines each expressing microRNAs directed against Klf4 (K4-1 or K4-2) or Klf5 (K5-1 or K5-2), as well as one cell line expressing microRNAs against LacZ. The proportions of fully undifferentiated colonies staining uniformly for AP are represented in black (Undiff), partially differentiated colonies showing heterogeneous AP staining are in gray (Mixed), and fully differentiated colonies are in white (Diff).

(D) Chimeric embryos made by aggregating indicated ESC lines with wild-type embryos dissected at 8.5 dpc. The presence of ESC-derived tissue is indicated by LacZ staining (blue). Areas where ES-derived cells have integrated with host embryos are indicated with arrows. Scale bars represent 1 mm. Images are representative of multiple examples of chimeric embryos.

(E) Quantitation of chimera experiments. The percentage of chimeric embryos displaying little or no integration (No Integration; white columns) of ESCs with the host cells or significant integration (Some Integration; black columns) of ESCs with the host embryo are plotted for Mbd3−/− ESCs expressing a scrambled siRNA (“Control,” n = 25), siRNA directed against Klf4 (“Klf4 RNAi,” n = 24) or Klf5 (“Klf5 RNAi,” n = 28), Klf4 RNAi cells rescued by expression of an RNAi-resistant Klf4 cDNA (“K4R,” n = 9), and Klf5 RNAi cells rescued by expression of an RNA-resistant Klf5 cDNA (“K5R,” n = 24). Scoring for contribution was performed blind to the ESC genotype.