Skip to main content
. 2014 Dec 31;4(1):103–113. doi: 10.1016/j.stemcr.2014.12.003

Figure 2.

Figure 2

Wnt Signaling Inhibitor Facilitates Generation of Naive Pluripotency Marker-Positive Cells from Mouse EpiSCs

(A) Western blots of β-CATENIN and active β-CATENIN in wild-type mouse EpiSC and mouse EpiSC with inducible E-cadherin transgene cultured with or without doxycycline (2 μg/ml) for 2 days (Dox−, Dox+). Cytoplasmic and nuclear fractions were analyzed. β-ACTIN and HISTON H3 were used as loading controls in cytoplasmic and nuclear fractions, respectively (left panel). Relative expression of β-CATENIN (upper-right panel) and proportion of β-CATENIN in cytoplasmic and nuclear fractions (right-lower panel) in each condition are shown.

(B) Western blotting analyses for phospho-STAT3 in mouse EB3DR ESCs (ESC), mouse EB3DR EpiSC (wild-type), and transgenic mouse EB3DR EpiSCs cultured with or without doxycycline (2 μg/ml) for 2 days (Dox−, Dox+). β-ACTIN was used as a loading control.

(C) Western blots of β-CATENIN and active β-CATENIN in mouse EpiSCs cultured with or without 5 nM IWP-2 for 2 days (DMSO, IWP-2). Cytoplasmic and nuclear fractions were analyzed. β-ACTIN and HISTONE H3 were used as loading controls in cytoplasmic and nuclear fractions, respectively.

(D) Timeline of naive-like cell induction using 5 nM IWP-2 and 1 μM PD0325901 in the presence of LIF. DMSO was used as control. Treatment was maintained for 7 and 14 days.

(E) Morphological observation of colonies derived from treated mouse EpiSCs (upper panel). Note the appearance of naive-like compact colonies (black arrowheads). Efficiency of generation of naive-like compact colonies at 14 days (lower panel). Efficiency of generation of naive-like compact colonies was calculated as the ratio of seeded cell number to the number of naive-like compact colonies (mean ± SEM of four independent experiments, ∗p < 0.05). Scale bar, 100 μm.

(F) FACS analysis of PECAM1 expression levels in mouse EpiSCs cultured with (blue line) or without (red line) IWP-2 for 7 and 14 days.

(G) Average of PECAM1-expressing cells associated with (F). The frequency of PECAM1-expressing cells increased significantly with IWP-2 treatment (mean ± SEM of three independent experiments, ∗∗p < 0.01, and ∗∗∗p < 0.001).

See also Figure S2.