Fig. 6: The integrin–actomyosin pathway regulates FGFR endocytosis.
From: ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification
a, GSEA with the RNA-seq data from H9 hESCs in low- and high-density cultures. GSEA was performed with three different gene sets: cell–substrate junction (left), cell–ECM interaction (middle) and integrin adhesome (right). b, Immunofluorescence assay for Integrin β1 and Paxillin in small and large H9 colonies. n = 9 regions for small, n = 13 regions for periphery, and n = 10 regions for centre. c, Immunofluorescence assay for pFAK and OCT4 in different densities of H9 hESCs. n = 69 regions. d,e, Immunofluorescence assay for pMLC in small (d) and large (e) H9 colonies. n = 30 regions. f, Immunofluorescence assay for FGFR1 and EEA1 in small H9 colonies treated with Blebbistatin (50 μM, 30 min). n = 10 regions. g, Time-course images for ERK-KTR in H9 hESCs treated with Blebbistatin (50 μM). ERK activity was measured by the ratio of the cytoplasmic over nuclear intensities (C/N ratio). n = 30 cells. h, Immunofluorescence assay for ETV4 in H9 hESCs treated with Blebbistatin (50 μM, 1 h). n = 241 cells for NC and n = 230 cells for Blebbistatin. i, Immunofluorescence assay for FGFR1 and EEA1 in large H9 colonies transduced with lentiviral vectors expressing constitutively active RhoAQ63L-GFP in a doxycycline-dependent manner. n = 25 regions. j, Immunofluorescence assay for PAX6 and Brachyury in small H9 colonies differentiated to either NE or ME cells with Blebbistatin (1 μM). NE differentiation: n = 37 regions, ME differentiation: n = 45 regions for NC and n = 38 regions for Blebbistatin. k, Immunofluorescence assay for PAX6 in H9 hESCs transduced with lentiviral vectors expressing ETV4-HA and differentiated to NE cells for 5 days with Blebbistatin (1 μM). n = 9 regions. l, Graphic summary of the mechanotransduction pathway regulating ETV4 expression and lineage fates. n is number of cells (g,h) or regions (b,c,e,f,i,j,k) pooled from three independent experiments. Two-sided Student’s t-test, ***P < 0.001, **P < 0.01, *P < 0.05; ns, not significant. Exact P values are presented in Supplementary Table 9. Scale bars: 10 µm (b,i), 25 µm (c,d,e,f,g), 50 µm (h,j,k). Numerical source data are available as Source data.
