Fig. 6: RHOA/ROCK-dependent actin remodeling is involved in YAP hyperactivation downstream of NUMB loss.

a Quantification of YAP nuclear/cytoplasmic ratio in BBN-WT and BBN-KO tumor cells treated with LatA (500 nM, 6 h), C3 transferase (3 µg/ml, 6 h), Y-27632 (ROCKi, 10 µM, 12 h), NSC-23766 (RACi, 10 µM, 12 h) or vehicle and co-stained for YAP, NUMB and DAPI. Graphs show the mean/field ± SEM, n = 11 fields/condition, from two independent experiments. ****, p < 0.0001; ***, p = 0.00064; not significant (ns) p-values are: p = 0.89, 0.91, 0.88 and 0.92, for BBN-WT cells treated with LatA, C3, ROCKi and RACi, respectively; p = 1 for BBN-KO cells treated with ROCKi, relative to matching controls by two-sided Tukey’s HSD test. Representative confocal images are shown in Supplementary Fig. 8. b Expression and phosphorylation status of the indicated Hippo pathway components in BBN-WT vs. BBN-KO cells, treated with C3 transferase (3 µg/ml, 6 h) or vehicle. Actin, loading control. Blots shown are representative of two independent experiments. c RT-qPCR for the indicated YAP transcriptional targets in BBN-WT vs. BBN-KO cells treated as in ‘b’. 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 C3, p = 0.043 (*); BBN-KO Veh, p = 0.0098 (**); Ctgf, BBN-WT C3, p = 0.068 (ns, not significant); BBN-KO Veh, p = 0.012 (*); Cyr61, BBN-WT C3; p = 0.017 (*); BBN-KO Veh, p = 0.017 (*). p-values by FDR-adjusted two-sided Welch’s t-test (C3- vs. vehicle-treated BBN-KO samples): Ankrd1, p = 0.01 (*); Ctgf, p = 0.006 (**); Cyr61, p = 0.027 (*). d Pull-down assay of activated RHOA in BBN-WT vs. BBN-KO cells. Blots are representative of two independent experiments. e Representative confocal fluorescence images of BBN-WT and BBN-KO tumor cells lentivirally transduced with RHOA-DN or empty vector (EV) and co-stained for RHOA, YAP and DAPI. Bars, 50 µm. Dashed line delineates a cluster of RHOA-DN overexpressing cells in BBN-KO cells. f Quantification of YAP nuclear/cytoplasmic ratio in BBN-WT- and BBN-KO cells treated as in ‘e’. Graphs show the mean/field ± SEM, n = 20 fields/condition, from two independent experiments. ****, p < 0.0001; ns, not significant (p = 1), relative to matching controls by two-sided Tukey’s HSD test. g Morphometric analysis of tumor BBN-MBO generated from cells described in ‘e’ (see legend to Fig. 4f). BBN-WT EV, n = 48; BBN-WT RHOA-DN, n = 48; BBN-KO EV, n = 37; BBN-KO RHOA-DN, n = 45, obtained from three independent experiments. ****, p < 0.0001; not significant (ns) p-values are: p = 1, 0.97, 0.91 and 0.82 for Area, Circularity, Roughness and Shape Complexity, respectively, relative to matching condition by FDR-adjusted pairwise two-sided Welch’s t-test. h Transwell Matrigel invasion assay of BBN-WT and BBN-KO cells treated with C3 transferase (3 µg/mL, 18 h) or vehicle. Number of invading cells/field expressed as the mean ± SEM of 8 microscope fields covering the migration area, from two independent experiments. ****, p < 0.0001; ns, not significant (p = 0.17), relative to matching controls by FDR-adjusted pairwise two-sided Welch’s t-test. Source data are provided as Source Data file.