Extended Data Fig. 11: Integrin β1 and Tensin 1 mediate viscoelasticity-specific mechano-cellular pathways involving YAP activation (Additional data to main Fig. 5).
From: Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver
a. Huh7-Cas9 cells were transfected with plasmids containing CRISPR guide RNA for TNS1 (sg-TNS1), or Integrin β1 (sg-Itg β1) or control sgRNA (NC), and cells after 24 h were embedded in low or high viscoelasticity hydrogels. RhoA GTPase activity in low/high viscoelasticity conditions and after TNS-1 or Integrin β1 KDs was tested by pull-down assays. Antibodies to active (non-phosphorylated), inactive YAP (phosphorylated), as well as to phosphorylated and total LATS1, were used, and analysed by immunoblotting. Representative images of 3 independent experiments. b. Quantification of GTP-RhoA/GAPDH protein levels from a (n = 3 each). c-e. Huh7 cells encapsulated in low or high viscoelasticity IPN hydrogels were incubated with ROCK (Y-27632, Abcam, 10 μM) or Myosin II inhibitors (Blebbistatin, Abcam, 50 μM). YAP activity was analysed by testing YAP-regulated target gene CTGF mRNA expression (c). Cell circularity was analysed by Image J (d), and cell proliferation was evaluated by Ki67 mRNA expression (e) (n = 3 each). Error bars represent mean ± s.e.m. n numbers refer to independent experiments. One-way ANOVA test was used followed by Tukey’s multiple comparison.
