Figure 1.

Effect of substrate stiffness on Treg induction. (A) Schematic of PA-gels coated with activating antibodies as Treg induction platform. (B) Mechanical testing via indentation indicates PA-gels with varying ratios of acrylamide and bis-acrylamide (n = 5 gels). (C) Treg induction on PA-gels with different stiffness (7 individual experiments and 2 replicates each with 4 donors, one-way ANOVA, ** p < 0.01, *** p < 0.001, **** p<0.0001). “+” corresponds to Dynabeads positive control, whereas “- “means unstimulated negative control without activating antibodies and cytokines. (D) Effect of blebbistatin on mechanosensing of Treg induction. 40 μM blebbistatin was introduced at the beginning of the culture (4 individual experiments and 2 replicates each, two-way ANOVA with Tukey’s multiple comparisons, ** p < 0.01, **** p<0.0001). (E) The correlation between activation and induction (data from 7.5 kPa, 55 kPa, and 140 kPa in (C), and the second-order polynomial fit was identified with the Extra sum-of-squares F test (α = 0.05). (F) Treg suppressive capacity represented as percent suppression with 1to1 ratio of Tregs to Tconvs (5 independent experiments, and the number of replicates depends on the number of cells obtained after sorting, Dy- Dynabeads, one-way ANOVA)