Fig 2: StemBond substrates support pluripotency.a Mean and standard deviation of mRNA expression of selected pluripotency genes on soft and stiff (mid AHA) substrates normalised to TCP. Cells were seeded on the substrates for 24 h in serum + LIF. The P-values show the significant differences in expression between soft and stiff substrates (one-way ANOVA). Expression was averaged over n = 4–6 independent experiments, points show the value of independent repeats. b Quantification of NANOG (left) and ESRRB (right) on different substrates from Western blots. Scatter plots show normalised band intensity from three independent experiments. Cells were cultured for 48 h in serum + LIF on fibronectin-coated TCP, soft and stiff hydrogels. Intensity were normalised to protein levels in 2i+LIF (2iL). Mouse embryonic fibroblasts (MEFs) were used as negative controls. P-values are from one-way ANOVA test. c (Left) Western blots for phospho-STAT3, phospho-ERK, total STAT3 and total ERK from cells grown on soft and stiff gels for 24 h in serum+LIF. GAPDH was used as loading control. (Right) Mean ± std of the ratio of intensity of soft/stiff after normalisation to GAPDH (n = 5 independent experiments). P-value computed using a one-way ANOVA. d Average percentage of reporter line Rex1 positive cells determined from flow cytometry histograms. Cells were cultured in serum + LIF on TCP (grey), and on soft (black) and stiff (red) high AHA substrates (n = 4 independent experiments). P-values from two-way ANOVA are indicated. Bars show mean ± standard deviation. Inset: Corresponding example of flow cytometry profiles. For each condition, histograms of three replicate samples were averaged and smoothed. Gating strategy show in Supplementary Fig. 4a. Source data are provided as a Source Data file.

Fig 3: Expression of Nanog in testis of various mammalian species as determined by RT-PCR.(A) Expression of Nanog in mouse testis of the Black 6 strain (BL/6) and the SV129 strain (SV129) (B) Expression of eGFP in testis of a TNG mouse; (C) Expression of NANOG in dog and pig testes; (D) Expression of NANOG in human testis; in all RT-PCR experiments, amplicons were of the expected sizes (See also Table 2) and products were identified by sequence analysis. Abbreviations: TNG = testis cDNA from Nanog eGFP reporter mouse; Cf = Canis familiaris, Ss = Sus scrofa, Hs = Homo sapiens; + = cDNA synthesis reaction performed with reverse transcriptase, − = minus reverse transcriptase control.

Fig 4: Models for the dynamic expression of NANOG in the epithelial cycle of murine (top) and canine (bottom) spermatogenesis.Each column represents the combination of different cell types that are present in seminiferous tubules at that specific stage. Roman figures = stage of the epithelial cycle, In = intermediate spermatogonia, B = type B spermatogonia, Pl = pre-leptotene stage, L = leptotene stage, Z = zygotene stage, P = pachytene stage, D = diplotene stage, 2nd = generation of secondary spermatocytes, 1–16 = steps in spermiogenesis. Cell types that express NANOG are outlined in red and cell types that do not express NANOG have black and grey symbols.

Fig 5: Immunoblot analysis.Composite image of immunoblot results for NANOG on lysates of mouse ES cells and dog and pig testes. On the right is a Benchmark protein ladder (Invitrogen) and the indicated molecular weights for each band. Abbreviations: mES = mouse embryonic stem cells, Cf = Canis familiaris, Ss = Sus scrofa.