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. Author manuscript; available in PMC: 2017 Feb 27.
Published in final edited form as: Nature. 2008 May 22;453(7194):519–523. doi: 10.1038/nature06968

Figure 4. CHIR99021 acts via inhibition of GSK3 to enhance ES-cell growth capacity and viability.

Figure 4

a, TOPFlash assay in ΔNLef1 stable transfectants. Results are shown as means and s.d. for three biological replicates. Non, N2B27 alone; CHIR, CHIR99021. b, Competitive growth assay for three clones of ΔNLef1 stable transfectants and Cre revertants (GFP+) in serum and LIF or in 3i. Results are shown as means and s.d. for four biological replicates. c, GSK3α/β-deficient DKO cells cultured in the indicated conditions for two passages. L, LIF. d, Phase-contrast and Oct4 immunostaining of DKO cells after eight passages in 1 μM PD0325901. Parallel cultures with addition of CHIR99021 were indistinguishable. e, ES cells constitutively expressing Nanog respond to CHIR99021 by enhanced self-renewal at low density in 3i compared with PS. f, ES cells self-renew in 2 μM PD0325901 plus CHIR99021. g, h, Diagrams of self-replication of the pluripotent state when inductive phospho-ERK signalling is either inhibited upstream by chemical antagonists (g) or counteracted downstream by LIF and BMP (h). Inhibition of GSK3 serves a key function in augmenting self-renewal when phospho-ERK (pERK) is suppressed by maintaining cellular growth capacity and additionally reinforcing suppression of neural commitment.