Fig. 4: Acquisition of feISC by CT treatment is dependent on YAP1 activation.
A WB analysis of PDO5 cells was collected after 72 h of 5-FU + Iri. treatment (from one out of three biologically independent experiments). B Representative images and quantification of YAP1 and ki67 staining in tumors derived from control (CT), IC20, and IC30-pre-treated PDOs implanted in nude mice. White arrows indicate cells with nuclear YAP1. DAPI was used as a nuclear marker (n = 9 independent regions examined). C RT-qPCR analysis of randomly selected feISC genes in control and 5-FU + Iri-treated PDO5 alone or plus 0.2 μM verteporfin (n = 2 biologically independent experiments). D RT-qPCR analysis of same genes in control and YAP1 KO PDO5 treated as in C (n = 2 biologically independent experiments). E, F Representative images of YAP1 E and SERPINH1 and S100A4 F in the indicated tumors at diagnosis or after neoadjuvant treatment (from 1 out of 6 biologically independent samples). G Representative image of an untreated human CRC identified as nuclear YAP1 high (done with all TMA samples, described in Supplementary Table S6). H, I Kaplan–Meier representation of disease-free H and overall I survival of CRC patients classified according to the H-score of nuclear YAP1. J Dose–response curves of IC20-pre-treated PDO5 and then treated for 3 days as indicated (n = 3 replicates examined, from one out of three biologically independent experiments). K Number of PDOs (upper panel) and diameter (lower panel) of PDO5 YAP1 KO after 72 hours of 5-FU + Iri. treatment and 2 weeks of washout. 300 cells/well were seeded (n = 6 wells examined for TICs and n = more than 50 spheres examined whenever possible for diameters, from three biologically independent experiments). L Comet assay in PDO5 YAP1 KO treated with 5-FU + iri. for 3 and 72 hours as indicated (n = >790 cells examined over three biologically independent experiments). For all applicable figure panels, data are mean ± SD, except for L (Tukey method for box plots), where boxes represent the central 50% of the data (from the lower 25th percentile to the upper 75th percentile), lines inside boxes represent the median (50th percentile), and whiskers are extended to the largest value less than the sum of the 75th percentile plus 1.5 IQR (the difference between the 25th and 75th percentile) or greater than the 25th percentile minus 1.5 IQR, and plot any values that are greater or lower than this as individual points. Significance (p) was calculated with one-way ANOVA, except for D and K by two-sided Student’s t test and for H and I by log-rank (Mantel–Cox) test. *p < 0.05, **p < 0.01, n.s., no significant. CT, control; IC20 and IC30, 5-FU + Iri. treatment leading to 20 and 30% cell death, respectively. Source data are provided as a Source Data file.
