Fig. 7: ROCK inhibition prevents YAP hyperactivation induced by loss of NUMB.

a Immunoblot of total and phosphorylated cofilin and YAP in BBN-WT vs. BBN-KO cells. Actin, loading control. Data are representative of two independent experiments. b Representative confocal images of BBN-WT and BBN-KO cells treated with vehicle or ROCKi, Y-27632 (10 µM, 12 h) and co-stained for phosphorylated MLC2 (p-MLC2) and DAPI. Bar, 50 µm. c Quantification of the experiment in ‘b’. Graphs show the relative intensity of p-MLC2 in BBN-KO vs. BBN-WT cells expressed as the mean/field ± SEM, n = 18 random fields/condition from two independent experiments. ****, p < 0.0001; not significant (ns), p = 0.59, relative to matching controls by two-sided Tukey’s HSD test. d RT-qPCR of the indicated YAP transcriptional targets in BBN-KO vs. BBN-WT cells treated with ROCKi Y-27632 (10 µM, 8 h) or vehicle. Graphs show the relative mean fold expression ± SEM from three independent experiments. p-values by FDR-adjusted two-sided one-sample t-test (vs. vehicle-treated BBN-WT): Ankrd1, BBN-WT ROCKi, p = 0.0013 (**); BBN-KO Veh, p = 0.0038 (**); Ctgf, BBN-WT ROCKi, p = 0.041 (*); BBN-KO Veh, p = 0.0047 (**); Cyr61, BBN-WT ROCKi, ns, not significant (p = 0.081); BBN-KO Veh, p = 0.00079 (***). In ROCKi- vs. vehicle-treated BBN-KO samples, p-values by FDR-adjusted two-sided Welch’s t-test are: Ankrd1, p = 0.022 (*); Ctgf, p = 0.0089 (**); Cyr61, p = 0.000026 (****). e Representative images of tumor BBN-MBOs generated from BBN-WT and BBN-KO cells treated with ROCKi (Y-27632, 10 µM). Bars, 100 µm. f Morphometric analysis of the indicated parameters in BBN-MBO cell treated as in ‘f’ (see legend to Fig. 4f); BBN-WT+Vehicle, n = 47; BBN-WT+ROCKi, n = 65; BBN-KO+Vehicle, n = 46; BBN-KO+ROCKi, n = 58, obtained from three independent experiments. ****, p < 0.0001; *, p = 0.023, 0.024 for Area and Roughness measures, respectively; not significant (ns), p = 0.07, 0.074 for Circularity and Shape Complexity measures, respectively, relative to matching condition by FDR-adjusted pairwise two-sided Welch’s t-test. g Transwell Matrigel invasion assay of BBN-WT and BBN-KO cells treated with ROCKi Y-27632 (10 µM, 18 h) or vehicle. Number of invading cells/field expressed as the mean ± SEM of 8 random microscope fields covering the entire migration area, from two independent experiments. ****, p < 0.0001; ***, p = 0.00023; not significant (ns), p = 0.33, relative to matching controls by FDR-adjusted pairwise two-sided Welch’s t-test. h GSEA enrichment plot of the EMT gene signature in BBN-KO vs. WT cells and BBN-KO cells treated with ROCKi 10 μM vs. vehicle. n = 2. ES, Enrichment Score; NES, Normalized Enrichment Score; p, two-sided permutation test p-value. Source data are provided as Source Data file.