Fig. 5: NUMB loss induces invasive phenotypes and YAP signaling activation in tumor urothelial cells.

a, b Morphology of 3D-Matrigel BBN-WT and BBN-KO tumor MBO exposed to 25 nM VP or vehicle for 7 days. Bar, 100 μm. Graphs in ‘b’ show the quantitation of the indicated morphometric parameters (see legend to Fig. 4f). BBN-WT+Veh, n = 47; BBN-WT + VP, n = 57; BBN-KO+Veh, n = 46; BBN-KO + VP, n = 45, obtained from three independent replicates. ****, p < 0.0001; ***, p = 0.0002; *, p = 0.023; ns, not significant (p = 0.42 for Circularity, 0.52 for Roughness and 0.87 for Shape Complexity), relative to matching condition by FDR-adjusted pairwise two-sided Welch’s t-test. c Transwell Matrigel invasion assay of BBN-WT and BBN-KO cells treated with VP (100 nM, 18 h) or vehicle. Number of invading cells/field expressed as the mean ± SEM of 8 microscope fields covering the entire migratory area, from two independent experiments. ****, p < 0.0001; not significant (ns), p = 0.54 by FDR-adjusted pairwise two-sided Welch’s t-test. d RT-qPCR for the indicated YAP transcriptional targets in BBN-WT vs. BBN-KO cells treated with VP (100 nM, 12 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 VP, p = 0.0098 (**); BBN-KO vehicle, p = 0.0098 (**); Ctgf, BBN-WT VP, p = 0.0062 (**); BBN-KO vehicle, p = 0.011 (*); Cyr61, BBN-WT VP, p = 0.017 (*); BBN-KO vehicle, p = 0.017 (*). p-values by FDR-adjusted two-sided Welch’s t-test (VP- vs. vehicle-treated BBN-KO samples): Ankrd1, p = 0.01 (*); Ctgf, p = 0.005 (**); Cyr61, p = 0.00093 (***). e GSEA enrichment plot of the YAP (left) and EMT (right) gene signatures in RNA-seq data from BBN-KO vs. BBN-WT cells (n = 3) and BBN-KO cells treated with VP vs. vehicle (n = 1). ES, Enrichment Score; NES, Normalized Enrichment Score; p, two-sided permutation test p-value. f Efficiency of YAP silencing in BBN-WT and BBN-KO lentivirally silenced for YAP (YAP-KD) or control-silenced (Ctr-KD). Actin, loading control. Results are representative of two independent experiments. g Morphology of 3D-Matrigel BBN-MBO from cells in ‘f’. Ctr-KD BBN-WT and BBN-KO MBO were also treated with vehicle or 25 nM verteporfin (VP). Bar, 100 μm. The red arrowhead indicates invading protrusions. h Quantification of morphometric parameters of BBN-MBO in ‘g’ (see legend to Fig. 4f). BBN-WT Ctr-KD+Veh, n = 48; BBN-WT Ctr-KD + VP, n = 40; BBN-WT YAP-KD+Veh, n = 42; BBN-KO Ctr-KD+Veh, n = 59; BBN-KO Ctr-KD + VP, n = 35; BBN-KO YAP-KD+Veh, n = 33, obtained from two independent experiments. ****, p < 0.0001; **, p = 0.008 for BBN-KO Ctr-KD + VP, p = 0.001 for BBN-KO YAP-KD+Veh; not significant (ns) p-values are: for BBN-WT Ctr-KD + VP, p = 0.83 (Area), 0.47 (Circularity), 0.77 (Roughness) and 0.59 (Shape Complexity); for BBN-WT YAP-KD+Veh, p = 0.83 (Area), 0.89 (Circularity), 0.67 (Roughness) and 0.16 (Shape Complexity), vs. matching condition, by FDR-adjusted pairwise two-sided Welch’s t-test. i Transwell Matrigel invasion assay of BBN-WT and BBN-KO cells in ’f’. Number of invading cells/field 24 h after seeding, expressed as the mean ± SEM of 8 fields from two independent experiments. **, p = 0.001 for BBN-KO Ctr-KD cells, p = 0.003 for BBN-KO YAP-KD cells; ns, not significant (p = 0.31), vs. matching controls by FDR-adjusted pairwise two-sided Welch’s t-test.