Fig. 3: 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.