Fig. 1: Mechanical microenvironments regulate ETV4 expression.
From: ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification
a, Schematic representation of high-density culture. Cell adhesive areas were reduced by matrigel-coated islands. b, Significant terms from InterPro protein ___domain analysis of 40 predicted upstream regulators of top 200 downregulated DEGs in crowded hESCs with high expression (>10 FPKM). A full list of the 40 proteins can be found in Supplementary Table 2. B-H, Benjamini-Hochberg. c,d, Representative images (c) and quantifications (d) of immunofluorescence assay for ETV1,4,5 and OCT4 in different densities of H9 hESCs. n = 40 cells for ETV1, n = 55 cells for ETV4, n = 45 cells for ETV5, n = 77 cells for OCT4. e, Western blots of ETV4 in different densities of H9 hESCs. n = 3 independent experiments. f, Immunofluorescence assay for ETV4 in scratched H9 hESCs. n = 40 cells for 0 h, n = 50 cells for 3, 6 and 12 h. Cell area was measured by dividing the total surface area by the number of cells. n = 9 regions for relative cell area. g, Immunofluorescence assay for ETV4 in MCF-7 cells. n = 30 cells for low density and n = 41 cells for high density. h, Immunofluorescence assay for ETV4 in H9 colonies on the cell stretching system. n = 60 cells. i, Immunofluorescence assay for ETV4 in MCF-7 cells on the cell stretching system. n = 59 cells for control and n = 46 reduced cells. j, Immunofluorescence assay for ETV4 and OCT4 in H9 hESCs on PDMS layers with different stiffnesses. ETV4: n = 80 cells for plastic and 15 kPa, n = 70 cells for 1.5 kPa; OCT4: n = 50 cells for plastic, n = 40 cells for 15 kPa and 1.5 kPa. k, Immunofluorescence assay for ETV4 in MCF-7 cells on PDMS layers with different stiffnesses. n = 45 cells. l, Immunofluorescence assay for ETV4 in hESCs after replating. n = 50 cells. n is number of cells (d,f,g,h,i,j,k,l) or regions (f) 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: 25 µm (c,g,h,i,j,k,l), 50 µm (f), 100 µm (a). Numerical source data and unprocessed gels are available as Source data.
