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Fig. S1. Conservation of Nanog sequence. ClustalW alignment of Nanog proteins. Shown is the sequence from the homeodomain to the C-terminus. NANOG, human; Nanog, mouse; cNanog, chicken; axNanog, axolotl; olNanog, medaka. Blue line indicates the caspase cleavage site (CCS); black line indicates the homeodomain (HD); orange line indicates the tryptophan (WR) domain.
Fig. S2. The tryptophan repeat (WR) domain is required for Nanog homodimers. Nanog forms dimers via its C-terminal WR domain. PCA results for analyses of Nanog ΔN, ΔHD and ΔC mutants. N, N-terminal; C, C-terminal; HD, homeodomain; CD2, terminal domain 2; WR, tryptophan repeat; Δ, deletion; Untr, untransfected; 1, hGL1 fused cDNA; 2, hGL2 fused cDNA. Values are normalized to untransfected controls (mean ± s.e.m.; n=3).
Fig. S3. Characterization of Nanog-expressing ESC lines. (A) Creation of constitutive and inducible Nanog dimers. Domains are depicted: CCS, caspase cleavage site; HD, homeodomain; CD1, C-terminal domain 1; WR, tryptophan repeat; CD2, C-terminal domain 2; ZIP, GCN4 leucine zipper; FKBPv, mutated FK506 binding protein domain. (B) PCA results for analyses of the interaction of ZIP and axNanog::ZIP. ZIP, GCN4 leucine zipper; untr, untransfected; 1, hGL1 fused cDNA; 2, hGL2 fused cDNA. Values are normalized to untransfected controls (mean ± s.e.m.; n=3). (C) PCA results for analyses of the self-interaction of FKBPv and axNanog::FKBPv with AP20187 treatment (FKBPv, mutated FK506 binding protein domain). AP20187 was added at the indicated concentration for 24 hours. Values are results of co-transfection of hGL1 and hGL2 fusions normalized to untreated values (mean ± s.e.m; n=3). (D) Gene expression analyses of puromycin-resistant ESC lines. Gene expression profile of ESC lines expressing Nanog (mean ± s.d.; n=3).
