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. 2016 Jan 26;26(2):171–189. doi: 10.1038/cr.2016.15

Figure 4.

Figure 4

SOX2 KD leads to enhanced non-neural lineage differentiation in hESCs due to activation of canonical Wnt signaling. (A) Western blot results of p-LRP6, LRP6, total β-catenin in H9 hESCs after SOX2/3 KD for 2 and 3 days with or without IWR1-e (10 μM). (B) Quantification of total β-catenin proteins on day 2 of SOX2/3 KD in hESCs shown in A by densitometry. (C) Western blot results of β-catenin, p-LRP6 and LRP6 in cytoplasm and nuclear extracts of hESCs on day 2 of SOX2/3 KD. OCT4 and GAPDH are loading controls for nuclear and cytoplasmic proteins, respectively. (D) Results of the 8× TOP/FOP luciferase reporter assay in hESCs on day 2 of SOX2/3 KD with or without IWR1-e (10 μM). (E) RT-qPCR results of Wnt pathway components in hESCs on day 3 of SOX2/3 KD. (F, G) RT-qPCR results of germ layer markers and Wnt targets (F) as well as pluripotency markers (G) on day 3 of ChIR99021 treatment (3 μM). (H-K) RT-qPCR results of mesoderm and trophectoderm markers (H), endoderm markers (I), EMT markers (J) and pluripotency markers (K) on day 3 of SOX2/3 KD in hESCs in the presence of vehicle, or noggin (200 ng/ml) or IWR1-e (10 μM).