Fig. 6: Substrate stiffness modulates the transcriptome of mESCs.

a Venn diagrams indicating the number of differentially expressed genes (p_adj < 0.05) in pair-wise comparisons. (Top) Number of genes regulated by substrate stiffness for low (n = 303), mid (n = 277) or high (n = 83) ECM tethering density. (Bottom) Genes regulated by ECM tethering on soft (51) and stiff (1) substrates. b Principal component analysis (PCA) computed on differentially expressed genes across all conditions (one point = one sample). Conditions are indicated by markers (stiffness) and colour-code (AHA concentration). c Heatmap of log₂fold change of expression for a selection of general pluripotency, naïve pluripotency and formative genes. All media conditions are compared to TCP control (log₂(hydrogel/TCP)). The vertical side of each triangle is proportional to the average log₂fc(soft/stiff) over all genes of a cluster. d Molecular functions and KEGG pathways that are enriched in the systematically modulated genes (n = 219). Dotted red lines represent the significant threshold (p_adj < 0.05). See also Supplementary Fig. 8d. e Cumulative sum of log₂fc for all genes (abs(log₂fc) > 0.2) belonging to pre-implantation (solid lines) or post-implantation (dotted lines) clusters. The diverging lines of the pre- and post-implantation genes indicate some systematic and opposing effect of substrate stiffness on those clusters (otherwise one would expect the solid and dotted lines would converge around zero cumulative log₂fc). Pie charts show the average percentage of significantly up/downregulated genes over all conditions (percentages for each condition given in Supplementary Fig. 8F). S: serum; S + L: serum+LIF; L: LIF; P: PD03; Pre:pre-implantation; Post: post-implantation. All time points were 48 h, except for “S + L 24 h”. log₂fc = log₂ fold change = log₂(soft/stiff). f (Top) Design of experiments for RNA-sequencing indicating the timing of the different steps and PD03 removal. Experiments performed in triplicate. (Bottom) Heatmaps of log₂ fold change in expression between soft and stiff substrates after PD03 removal. (Left) Top 50 activated genes and (right) Top 50 suppressed genes. The genes listed are those that are co-regulated after inhibitor removal on both soft and stiff and show the largest fold change on stiff substrates when compared to control in 2i+LIF.