Fig. 5: The integrin-β1–TNS1–YAP axis mediates viscoelasticity-specific mechanocellular pathways for HCC cell invasion.
From: Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver
a, Schematics of the tuneable viscoelasticity IPNs of alginate (blue) and reconstituted basement membrane matrix (green) 3D hydrogels. Lowering the molecular mass of alginate cross-linked by calcium (red) decreases the network connectivity (arrows) to increase viscoelasticity. The diagram was adapted from ref. 16, under a Creative Commons licence CC BY 4.0. b–g, Cell proliferation in low- or high-viscoelasticity hydrogels was analysed using EdU assays (imaging (b) and quantification (e)). c,f, YAP activation was analysed using antibodies against active YAP (imaging (c) and quantification (f); the arrowheads denote enlarged areas). d, The formation of invadopodia-like structures after transfection (Clontech-N1, containing human TKS5-mNeonGreen), and immunofluorescence analysis of MT1-MMP (red) using Airyscan microscopy. g, Cell circularity analyses (ImageJ; n = 5 gels). Scale bars, 50 μm (b), 20 μm (c) and 10 μm (d). h–j. Huh7 cells in low- or high-viscoelasticity hydrogels were incubated with control IgG or integrin β1 (ITGB1) blocking antibodies. Cell proliferation (h; EdU), YAP target CTGF mRNA (i) and cell circularity (j, n = 4) were analysed. k, TNS1 mRNA expression in cells in low- or high-viscoelasticity hydrogels. n = 3. l,m, PLAs depict direct binding between TNS1 and integrin β1 (ITGB1) in high-viscoelasticity hydrogels (l; scale bar, 10 μm). m, The PLA signal was analysed (30 cells in 5 gels per group, n = 5, each). n–p, Huh7-Cas9 cells were transfected with plasmids containing CRISPR guide RNA for TNS1 (sgTNS1), integrin β1 (sgITGB1) or control sgRNA (NC) and embedded in low- or high-viscoelasticity hydrogels. The proliferation (n), YAP activation (o) and cell circularity (p) were analysed. n = 5 each. q, Schematic of TNS1, which functions as a key component of the ECM mechanosensor complex by binding to integrin β1 in high-viscoelasticity ECM. The diagram was created using BioRender. Data are mean ± s.e.m. n values refer to independent experiments. Statistical analysis was performed using two-tailed unpaired t-tests (e–g and k) and one-way ANOVA followed by Tukey’s multiple-comparison test (h–j and n–p).
