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. 2016 Dec 1;143(23):4368–4380. doi: 10.1242/dev.138982

Fig. 3.

Fig. 3.

Acquisition of mESC-like signaling pathways in LIF-3i-reverted hPSCs. (A) Stabilization of nuclear P-STAT3 and cytoplasmic activated (activ) β-catenin in LIF-3-reverted hPSCs. Nuclear (Nu), cytoplasmic (Cy) and total (Tot) fractions are shown for H9 hESC, E5C3 sa-MP-iPSC and C1.2 fibro-iPSC lines. TBP and actin are protein loading controls. +, LIF-3i; −, primed culture. (B) Inhibition/proliferation assays of sa-MP-iPSC line E5C3. Shown are mESC signaling pathways [e.g. LIF/JAK/STAT, LIF-receptor/gp130, CREB, BMP4 (dorsomorphin) and PI3K]. Cumulative proliferations of SSEA4+ TRA-1-81+ E5C3 cells were measured after 12 days of culture in the presence of indicated inhibitors, normalized to LIF-3i-alone conditions (at 100%). (C) Western blots of key WNT components AXIN1 and activated β-catenin in both nuclear and cytoplasmic fractions in primed (−) versus LIF-3i reverted (+) hPSC cultures. (D,E) Confocal microscopy of WNT proteins in indicated primed (bFGF) versus LIF-3i-reverted hPSC lines. Activated β-catenin is shown to be distinctly sequestered outside of the nuclear compartment, whereas uniform expression of OCT4 was localized strictly within nuclei. Scale bars: 20 µm.