Fig. 8: Loss of NUMB triggers RHOA/ROCK-dependent YAP hyperactivation in human RT4 BCa cells.

a Expression of NUMB and the indicated Hippo pathway components in control- (Ctr-KD) and NUMB-silenced (NUMB-KD) RT4 cells. GRP94, loading control. Data are representative of two independent experiments. b Quantification of YAP nuclear/cytoplasmic ratio in Ctr-KD and NUMB-KD RT4 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 endogenous YAP and DAPI. Representative confocal images are shown in Supplementary Fig. 10a. Data are shown as the mean/field ± SEM, n = 8 fields/condition, from two independent experiments. ****, p < 0.0001; not significant (ns) p-values are: p = 0.98, 0.98, 1 and 1, for RT4 Ctr-KD cells treated with LatA, C3, ROCKi and RACi, respectively, p = 0.33 for RT4 NUMB-KD cells treated with RACi, relative to matching controls by two-sided Tukey’s HSD test. c Expression of NUMB, and total and phosphorylated YAP and MST1 in Ctr-KD vs. NUMB-KD RT4 cells treated with vehicle or C3 transferase (3 μg/mL, 6 h). Actin, loading control. Data are representative of two independent experiments. d Immunoblot of NUMB, total and phosphorylated cofilin, and phosphorylated MLC2 (p-MLC2), in Ctr-KD vs. NUMB-KD RT4 cells. Actin, loading control. Blots are representative of two independent experiments. e RT-qPCR of the indicated YAP transcriptional targets in NUMB-KD vs. Ctr-KD RT4 cells treated with C3 (3 µg/ml, 6 h) (left) or ROCKi Y-27632 (50 µM, 8 h) (right), 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 RT4 Ctr-KD): ANKRD1, Ctr-KD C3, not significant (ns, p = 0.076); NUMB-KD Veh, p = 0.042 (*); CTGF, Ctr-KD C3, ns (p = 0.36); NUMB-KD Veh, p = 0.03 (*); CYR61, Ctr-KD C3, p = 0.011 (*); NUMB-KD Veh, p = 0.007 (**) (left); ANKRD1, Ctr-KD ROCKi, ns (p = 0.263); NUMB-KD Veh, p = 0.013 (*); CTGF, Ctr-KD ROCKi, ns, (p = 0.7); NUMB-KD Veh, p = 0.034 (*); CYR61, Ctr-KD ROCKi, ns (p = 0.52); NUMB-KD Veh, p = 0.009 (**) (right). p-values by FDR-adjusted two-sided Welch’s t-test: C3- vs. vehicle-treated NUMB-KD (left), ANKRD1, p = 0.03 (*); CTGF, p = 0.0072 (**); CYR61, p = 0.0022 (**); ROCKi- vs. vehicle-treated NUMB-KD (right): ANKRD1, p = 0.0045 (**); CTGF, p = 0.0016 (**); CYR61, p = 0.00057 (***). f Enrichment of the EMT gene signature by GSEA in NUMB-KD RT4 cells treated with ROCKi vs. vehicle as in ‘e’. n = 1. ES, Enrichment Score; NES, Normalized Enrichment Score; p, two-sided permutation test p-value. g Morphology of organoids from stable Ctr-KD vs. NUMB-KD RT4 cells treated with vehicle (Veh), verteporfin (VP, 25 nM) or ROCKi Y-27632 (10 µM). The red arrow points to invasive protrusions in NUMB-KD RT4 cells. Bar, 100 µm. h Morphometric analysis of the experiment in ‘g’ (see legend to Fig. 4f). Ctr-KD+Vehicle, n = 122; Ctr-KD + VP, n = 77; Ctr-KD+ROCKi, n = 59; NUMB-KD+Vehicle, n = 68; NUMB + VP, n = 54; NUMB+ROCKi, n = 50, obtained from two independent experiments. ****, p < 0.0001; ***, p = 0.00032; **, p = 0.006; *, p = 0.024 (Circularity), 0.034 (Roughness), 0.034 (Shape Complexity); not significant (ns) p-values are: p = 0.2, 0.37 and 0.19, for Circularity, Roughness and Shape Complexity, respectively, for RT4 Ctr-KD cells treated with VP; p = 0.37, 0.17 and 0.12, for Circularity, Roughness and Shape Complexity, respectively, for RT4 Ctr-KD cells treated with ROCKi, relative to matching condition by FDR-adjusted pairwise two-sided Welch’s t-test. i Transwell Matrigel invasion assay of Ctr-KD and NUMB-KD RT4 cells treated with vehicle, VP (100 nM), C3 (3 µg/mL) and ROCKi Y-27632 (10 µM) for 48 h. Number of invading cells/field expressed as the mean ± SEM of 8 microscope fields from two independent experiments. **, p = 0.003, 0.002 and 0.003, for RT4 NUMB-KD cells treated with vehicle, VP and C3, respectively; *, p = 0.046; not significant (ns) p-values are: p = 0.091, 0.99 and 0.091, for RT4 Ctr-KD cells treated with VP, C3 and ROCKi, respectively, vs. matching condition by FDR-adjusted pairwise two-sided Welch’s t-test. Source data are provided as Source Data file.