Extended Data Fig. 9: Cell crowding blocks FGFR endocytosis.
From: ETV4 is a mechanical transducer linking cell crowding dynamics to lineage specification
a, Immunofluorescence assay for FGFR1 and EEA1 in small and large H9 colonies. Arrow heads indicate apical localization of FGFR1 proteins. n=6 regions for Small, n=8 regions for Large. b, Immunofluorescence assay for FGFR1 and E-cadherin in the centre of large H9 colonies. n=8 regions. c, Validation of TD0 (500 nM) in human dermal fibroblasts expressing ERK-KTR. n=22 cells pooled from two independent experiments. d, Immunofluorescence assay for FGFR1 and EEA1 in large H9 colonies treated with TD0 (500 nM, 1h). n=9 regions pooled from two independent experiments. e, Immunofluorescence assay for EEA1 in H9 hESCs treated with Pitstop2 (50 μM, 1h) or Dynasore (100 μM, 1h). n=28 regions for NC, n=12 regions for Pitstop2, n=16 regions for Dynasore pooled from two independent experiments. f, Immunofluorescence assay for FGFR1 and EEA1 in H9 hESCs treated with Dynasore (100 μM, 1h). n=8 regions pooled from two independent experiments. g, Time-course images for ERK-KTR in H9 hESCs treated with Dynasore (100 μM). ERK activity measured by the ratio of the cytoplasmic over nuclear intensities (C/N ratio). n=46 cells. h, Immunofluorescence assay for ETV4 in H9 hESCs treated with Dynasore (100 μM, 1h). n=140 cells pooled from two independent experiments. i, Immunofluorescence assay for ETV4 in H9 hESCs transduced with lentiviral vectors expressing KRASG12V together with GFP and treated with Pitstop2 (50 μM, 30 min). n=45 cells. j, Immunofluorescence assay for PAX6 in H9 hESCs transduced with lentiviral vectors expressing FGFR1 shRNAs together with GFP and differentiated to NE cells for 5 days. n=10 regions. n = number of cells (g and i) or regions (a,b, and j) 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: 10 µm (e,f, and j), 15 µm (a), 25 µm (d,g,h, and i), 50 µm (b), 100 µm (c). Numerical source data are available in Source data.
