Fig. 2: Catalytic activities of TET DNA demethylases are indispensable for the maintenance of pluripotency during dormancy.
From: TET activity safeguards pluripotency throughout embryonic dormancy
a, Proliferation curves and brightfield images of wild-type and Dnmt3a/b DKO ES cells (devoid of de novo methyltransferase activity) treated with the mTOR inhibitor INK128 for 120 h. Data are from two biological replicates. Individual data points are shown; lines denote the mean. b, Same as in a for Tet1/2/3flox/flox versus Tet1/2/3 TKO iPS cells30. Tet TKO cells lose pluripotent colony morphology over time under dormancy conditions. c, Rescue of Tet TKO dormancy defect via overexpression of wild-type, but not catalytic-dead (cd), Tet1 or Tet2. The catalytic-dead mutations can be found in Methods and Extended Data Fig. 3d. Images are representative of two biological replicates. d, Alkaline phosphatase staining of an independently generated, feeder-independent Tet1/2 DKO ES cell line in normal and mTORi conditions. See Extended Data Fig. 3e–g for details of the deletions and accompanying proliferation curves. Tet1/2 DKO ES cells lose pluripotent colony morphology and marker (alkaline phosphatase) expression during mTORi treatment. The rightmost images are magnifications of the asterisk-marked colonies. Images are representative of two biological replicates. e, Rescue of Tet TKO dormancy defect in the absence of DNMT activity. Wild-type or Dnmt TKO ES cells were treated with the TET inhibitor (TETi) Bobcat339 with or without mTORi. TETi-treated cells are depleted specifically under mTORi treatment in wild-type but not Dnmt TKO ES cells. Individual data points are shown; lines denote the mean. f, Flow cytometry analysis of SSEA1 expression levels (a pluripotency marker) in wild-type and Tet1/2 DKO cells in normal and mTORi conditions. Left: overlays of SSEA1 expression at 0 h versus 96 h in wild-type or Tet1/2 DKO cells. Right: stacked bar plots showing quantification of SSEA1 expression levels at all quantified time points. All flow cytometry plots are shown in Extended Data Fig. 4a. Data from two biological replicates are shown.
