Fig. 5: ERM proteins determine the mechanical differences between LGR5+ and LGR5- cells.

a Normalized Enrichment Scores (NES) from enrichment analysis ROAST-GSA for selected GO cellular component (GOCC) gene sets in LGR5-, LGR5^med and LGR5+ cells. p values are listed in Supplamentary Table 1. Data obtained from two independent replicates (at least 20,000 cells per condition). b Relative expression of Lgr5, Krt20 and ERM proteins for sorted cells. Data are represented as the mean ± s.d. from nine independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. c Levels of ERM and phospho-ERM proteins in sorted PDO cells with quantification of band intensity normalized to GAPDH from two independent experiments. d Scheme of the iMC-linker. e mRNA levels of ERM proteins in wild type and shRNA ERM silenced organoids. Data are represented as the mean ± s.d. from two independent experiments. f LGR5+ cells treated with doxycycline to induce the iMC-linker expression, untreated (no doxy) and LGR5- ERM silenced cells on 3 kPa gel substrates. Representative images of two independent experiments. Scale bar, 50 μm. g Cell roundness for sorted cells seeded on 3 kPa gels. Data are represented as the mean ± s.d. of n ≥ 58 cells/condition from two independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. h Representative images of LGR5-, LGR5+ and iMC-linker expressing LGR5+ cells under confinement. Scale bar, 10 μm. i Percentage of dying/dead, polarized blebs, rounded blebs and no blebs in LGR5-, LGR5+ and iMC-linker expressing LGR5+ cells. Data are represented as the mean ± s.d. of percentages from four independent experiments (n ≥ 119 cells/subgroup). Statistical significance was determined using two-way analysis of variance, followed by a Šidák multiple-comparison test. j Migration velocity of LGR5^high, LGR5^low, iMC-linker expressing LGR5^high clusters and LGR5^low clusters with ERM silencing. Data are represented as the mean ± s.d. of n ≥ 46 clusters/condition from two independent experiments. Statistical significance was determined using Shapiro-Wilk normality test, followed by a Kruskal−Wallis multiple-comparison test. k Time lapse of LGR5^high, LGR5^low, iMC-linker expressing LGR5^high and ERM silenced LGR5^low clusters seeded on Collagen-I coated 3 kPa gels. Representative images of two independent experiments. Scale bars, 50 μm. Source data are provided as a Source Data file.