Fig. 3: Cell-crowding-induced ETV4 inactivation derepresses the NE fate.
From: ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification
a, Immunofluorescence assay for PAX6, SOX2, Brachyury in differentiated H9 hESC colonies. n = 7 colonies. b, Immunofluorescence assay for PAX6 and Brachyury in H9 hESCs cells differentiated to either NE or ME cells. n = 25 regions for NE, n = 40 regions for ME. c, Quantitative analysis of the relationship between the diameters of ETV4-low areas in undifferentiated H9 hESC colonies and the diameters of PAX6+ areas in NE-differentiated colonies. n = 168 colonies for ETV4 and n = 92 colonies for PAX6. d, Immunofluorescence assay for PAX6 in large H9 colonies transduced with lentiviral vectors expressing ETV4-HA and differentiated to NE cells for 5 days. n = 15 regions. e, Immunofluorescence assay for PAX6 in small H9 colonies transduced with lentiviral vectors expressing ETV4 shRNAs together with GFP and differentiated to NE cells for 5 days. n = 10 regions. f, Volcano plot showing DEGs in H9 hESCs after ETV4 KD. The red and blue dots indicate upregulated and downregulated genes, respectively, with cutoff values for DEGs: log2(fold change) < −0.5 or > 0.5, adjP < 0.05. Full list of DEGs can be found in Supplementary Table 3. g, Immunofluorescence assay for PAX6 and Brachyury in small H9 colonies differentiated to either NE or ME cells with pan MMP inhibitors, GM6001 (5 μM) and BB94 (2 μM). NE differentiation: n = 21 regions for GM6001, n = 20 regions for BB94; ME differentiation: n = 38 regions for GM6001, n = 52 regions for BB94. h, Immunofluorescence assay for PAX6 in shETV4-expressing H9 hESCs transduced with lentiviral vectors expressing MMP14 together with GFP and differentiated to NE cells for 5 days. n = 9 regions. n is number of colonies (a,c) or regions (b,d,e,g,h) 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 (e), 25 µm (h), 50 µm (a,d,g), 100 µm (b). Numerical source data are available in Source data.
