Patient-derived cancer HCOs exhibit different sensitivity to centrosome loss independently of p53.

A) The indicated cancer HCO lines (control or p53 KO) were grown from single adult stem cells in the presence of DMSO or 0.5 μM centrinone B for 8 days. Organoids were then fixed, stained with DAPI (blue) and phalloidin (red) and imaged. Merged maximum intensity projection images are shown. B) The areas of individual organoids were quantified in the merged maximum intensity projection images from (A) and presented in the graph as relative values normalized to the respective DMSO control; every dot represents an organoid (n = 3, ***P<0.001, One-way ANOVA with Bonferroni post-hoc). C) p53 mutation status in the three cancer HCOs from the whole exome sequencing data. D) Western blot analysis of p53 and loading controls (GAPDH and actin) in lysates prepared from the indicated control and p53 KO cancer HCO lines. E) Quantification of (D). Protein levels from three independent experiments were quantified and presented in the graph (n = 3, **P<0.01, ***P<0.001). F) High-resolution maximum intensity projections of pericentrin (centrosome) and DNA (DAPI) immunofluorescence staining in POP-112 organoids treated with DMSO or 0.5 μM centrinone B for 8 days. G) The number of pericentrin foci and nuclei were automatically identified in z-stack sections spanning the entire organoid. The foci to nucleus ratio was determined for four organoids in each condition (***P<0.001). H) POP-112 organoids were grown from single adult stem cells in the presence of DMSO (0) or the indicated centrinone B concentrations for 8 days. Organoid areas were quantified as in (B). Relative average organoid area from three independent experiments is presented in the graph (n = 3, **P<0.01, ***P<0.001, all other treatments compared to DMSO are not significant, One-way ANOVA with Bonferroni post-hoc). Scale bars are (A) 500 μm and (F) 25 μm.