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. 2017 Jun 9;8(6):1645–1658. doi: 10.1016/j.stemcr.2017.05.014

Figure 5.

Figure 5

Klf4 Represses the Blimp1-Positive State in ESCs Downstream of STAT3

(A) Percentage of Blimp1+ ESCs in 2i/L with or without the JAK kinase inhibitor or the phosphatidylinositol 3-kinase (PI3K) inhibitor.

(B) Percentage of Blimp1+ ESCs in 2i with forced expression of the indicated LIF target gene (A and B). n = 3 biological replicates each assayed in technical triplicate; error bars, SEM.

(C) Immunofluorescence staining of Blimp1-GFP and KLF4 in ESC colonies showing mutually exclusive expression. Scale bar, 50 μm.

(D) Immunofluorescence showing knockout (KO) of Klf4 in independent ESC lines and undetectable effects on pluripotency gene NANOG. Scale bars, 50 μm.

(E) qRT-PCR analysis of gene expression in Klf4 KO ESCs. n = 3 biological replicates each assayed in technical triplicate.

(F) FACS analysis showing a significant increase in the Blimp1+ fraction in Klf4 KO ESCs.

(G) Western blot showing KLF4 transgene rescue to WT-equivalent levels (−) or overexpression (+) in Klf4 KO cells.

(H) Re-expressing KLF4 re-imposes repression of the Blimp1+ ESC state in a dose-dependent manner. n = 3 biological replicates each assayed in technical triplicate; error bars, SEM.