Fig. 4: NUMB loss induces invasive phenotypes and YAP signaling activation in normal urothelial cells.

a Left, Representative confocal IF images of endogenous YAP expression in FFPE sections of WT and NUMB-KO mouse bladder organoids (MBOs) grown in 3D-Matrigel. Nuclei are stained with DAPI. Bars, 100 μm; Mag., 50 μm. The % of YAP-positive cells (middle) and the mean nuclear YAP intensity (arbitrary units, A.U.) (right) are expressed as mean ± SEM, n = 4 fields/condition from one experiment, representative of two independent experiments. *, p = 0.011 (left), 0.017 (right) by two-sided Welch’s t-test. b RT-qPCR for the indicated YAP transcriptional targets in WT- and NUMB-KO MBO grown in 3D-Matrigel in the presence of vehicle (Veh) or 200 nM verteporfin (VP) for 48 hr. 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 MBO-WT): Ankrd1, MBO-WT VP, p = 0.00086 (***); MBO-KO Veh, p = 0.0083 (**); Ctgf, MBO-WT VP, p = 0.13 (ns, not significant); MBO-KO Veh, p = 0.041 (*); Cyr61, MBO-WT VP, p = 0.0017 (**); MBO-KO Veh, p = 0.0032 (**). p-values by two-sided Welch’s t-test (VP- vs. vehicle-treated MBO-KO samples): Ankrd1, p = 0.004 (**); Ctgf, p = 0.013 (*); Cyr61, p = 0.00011 (***). c GSEA enrichment plot of the EMT gene signature in RNA-seq data from WT- vs. NUMB-KO MBO cells (n = 6 for each condition) and NUMB-KO MBO treated with 200 nM VP vs. vehicle for 48 hr. (n = 1 for each condition). ES, Enrichment Score; NES, Normalized Enrichment Score; p, two-sided permutation test p-value. d Representative images of 3D-Matrigel WT- and NUMB-KO MBO grown in the presence of vehicle (Veh) or 25 nM verteporfin (VP) for 10 days. Bar, 100 μm. e Frequency of occurrence of normal and aberrant multiacinar morphology in MBO in ‘c’. MBO-WT+Veh, n = 219; MBO-WT + VP, n = 119; MBO-KO+Veh, n = 174; MBO-KO + VP, n = 189, obtained from three independent experiments. ****, p < 0.0001; ns, not significant (p = 0.35), relative to matching condition by FDR-adjusted pairwise two-sided Fisher’s t-test. f Analysis of morphometric parameters (Area, Circularity, Roughness, and Shape Complexity) of WT vs. NUMB-KO MBO treated as in ‘d’. Values are reported, in this and other relevant panels in the figure, as boxplots delimited by 25th and 75th percentiles and showing the median (horizontal line) and the mean (X). The whiskers span from the smallest and largest data values within a 1.5 interquartile range. MBO-WT+Veh, n = 219; MBO-WT + VP, n = 119; MBO-KO+Veh, n = 174; MBO-KO + VP, n = 189, obtained from three independent experiments. ****, p < 0.0001; *, p = 0.027; ns, not significant (p = 0.058 for Area, 0.15 for Circularity and 0.9 for Shape Complexity), relative to matching condition, by FDR-adjusted pairwise two-sided Welch’s t-test. Source data are provided as Source Data file.