Ext Data Fig 3

A) Unsupervised hierarchical clustering of differentially expressed genes from bulk RNA sequencing. Cells from soft and stiff viscous gels are compared to cells in non-adherent tissue culture with or without calcium control. The calcium control are cells exposed to free calcium released from an empty hydrogel. Markers were separated into 11 clusters associated with patterns of gene expression. B) Clusters 5, 6, 7, 8, and 11 were associated with genes upregulated in stiff viscous conditions. These markers were put into the ENCODE ChIP-Seq database, which identified STAT3 as one of the top ranked transcription factors involved in regulating these genes. C) Flow cytometry of HLA-DR MFI and CD11b+CD11c+ cells (gated on live-HLA-DR+) with blocking anti-IL6R antibody (1ug/ml), n=3 biological replicates. D) Normalized gene expression of NFKBIZ at Day 0 prior to encapsulation and after 3 days in viscous and elastic gels of soft (0.75 kPa) and stiff (7.5 kPa) elastic modulus. Data points indicate biological replicates from n=2 donors for viscous and elastic gels, and n=1 donor for day 0 control. Experiments were repeated with at least two donors.