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. 2016 Oct 3;113(42):E6382–E6390. doi: 10.1073/pnas.1613849113

Fig. 1.

Fig. 1.

Naïve hESCs have active endogenous Wnt/β-catenin signaling independent of the presence of CHIR. (A) qRT-PCR results of AXIN2 and TROY expression in naïve (2iLIF) or primed (AF) ELF1 hESCs (Top); naïve ELF1 hESCs cultured in media with vehicle control (DMSO), XAV939 (XAV, 5 µM), or IWP2 (2 µM) for 3 d (Middle); or naïve ELF1 hESCs in media with 2iLIF for 3 d after transfection with negative control (Ctrl) siRNAs, CTNNB1 siRNAs, and AXIN1/2 siRNAs (Bottom). Cells in all conditions were cultured on Matrigel with MEF-CM for 3 d (mean ± SEM of n = 3 biological replicates; *P < 0.05 by t test). (B) Representative fluorescence images of BAR-Venus expression of ELF1-BAR hESCs grown on MEFs in media with CHIR or without CHIR (w/o CHIR) for three passages. (Scale bar, 100 µm.) (C) Representative plots of flow cytometry for Tra1-60 and CD9 expression in ELF1 hESCs cultured in media with or w/o CHIR for three passages (last passage on Matrigel) (mean ± SEM of n = 3 biological replicates; *P < 0.05 by t test). (D) Representative images and flow cytometry histograms of BAR-Venus expression in naïve ELF1-BAR hESCs cultured on MEFs in media with or w/o CHIR and with the indicated small molecules for 3 d. At the end of day 3, cells were lifted and passaged into new wells with MEFs and cultured with the indicated small molecules from days 4–6 (single data point representative of n = 2 biological replicates). (Scale bar, 100 µm.)