(A) Western blot analysis of
Gsk3α−/−,
Gsk3β−/−, and
Gsk3α/β DKO ESCs.
Gsk3α/β DKO ESCs were generated by knocking
out Gsk3α in
Gsk3β−/− ESCs.
β-Catenin phosphorylation at S33/S37/T41 was examined in the indicated
ESC lines using a phospho-β-catenin antibody from Cell Signaling
Technology (no. 9561, lot 12). The same antibody was used to examine
phosphor-β-catenin throughout the study.
(B) TopFlash assay in wild-type and GSK3 mutant ESCs treated with 3 μM
CHIR for 8 hr. Non, no treatment control. Data represent means ± SD of
three biological replicates. **p < 0.01;
***p < 0.001.
(C) Representative images of E14TG2a ESCs cultured in LIF/serum or LIF/serum
+ 3 μM CHIR for 5 days. Scale bars, 100 μm.
(D) Representative images of different GSK3 mutant ESC lines cultured in
LIF/serum for 5 days.
Gsk3β−/− ESCs formed
both compact (arrows) and flat colonies. Scale bars, 100 μm.
(E) Quantification of flat and compact colonies formed from the indicated ESC
lines. ESCs were plated onto six-well plates at clonal density and cultured in
LIF/serum (if not specified) or LIF/serum + 3 μM CHIR (E14
+ CHIR) for 7 days. E14, E14TG2a ESCs. Data represent means ± SD
of three biological replicates.