Extended Data Fig. 3: ETV4 and YAP exhibit distinct sensitivities to changes in cell density in hESCs.
From: ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification
a, The statistical analysis of cell shape index and relative cell area of ETV4-high and ETV4-low cells in large H9 colonies. n=90 cells. b, Gene set enrichment analysis (GSEA) with the RNA-seq data from H9 hESCs in low and high density cultures. GSEA was performed with 2 different gene sets: Hallmark_Glycolysis, Hallmark_Hypoxia from Molecular Signatures Database (MSigDB). c, Immunofluorescence assay for Hypoxyprobe in small and large H9 colonies. n=9 regions. d,e, Glucose uptake assay in small and large H9 colonies. n=25 regions for 37°C (d), n=31 regions for 4°C (d), and n=30 regions for (e). f, Immunofluorescence assay for YAP in large H9 colonies. YAP activity was measured by the ratio of the nuclear over cytoplasmic intensities (N/C ratio). n=5 colonies. g, Immunofluorescence assay for YAP/TAZ and OCT4 in large H9 colonies. n=80 cells pooled from two independent experiments. h,i, Validation of lentiviral vectors expressing YAP shRNAs in H9 hESCs by qPCR (h) and immunostaining (i). n=3 samples for qPCR (h) and n=50 cells for immunostaining (i). j, Quantification of cells in M phase in large H9 colonies. n=20 regions. k, Immunofluorescence assay for Ki67 in large H9 colonies. n=40 cells pooled from two independent experiments. l, Immunofluorescence assay for ETV4 and EdU in large H9 colonies. n=6 regions. m, Crystal violet staining in H9 hESCs transduced with lentiviral vectors expressing YAP shRNAs. n = number of cells (a and i), colonies (f), or regions (c,d,e,j, and l) pooled from three independent experiments. Two-sided Student’s t-test, ***P < 0.001, **P < 0.01, *P < 0.05. Exact P values are presented in Supplementary Table 9. Scale bars: 25 µm (a,c,d,e,g, and i), 50 µm (l), 100 µm (k). Numerical source data are available in Source data.
