Fig. 5. The Trp53/miR-34 axis is disrupted in the Wnt-independent organoids.
a Organoids immunostained for p53 (green) and β-catenin (red). DAPI (blue) was used to stain for DNA. Image representative of three independent replicates. Scale bars: 10 μm. b Wnt-independent organoids grow in the presence of Nutlin-3a in the medium. Image representative of three independent replicates. Scale bars: 1 mm. c Western blot to verify knockout of Trp53 in organoids after transduction with AAV-Cre. Image representative of two independent replicates. d Organoids from Trp53flox/flox mice grow in the presence of Nutlin-3a and absence of Wnt and CHIR99021 only when transduced with AAV-Cre. Image representative of two independent replicates. Scale bars: 100 μm. e RT-qPCR data showing expression of pri-miR-34 relative to TUBB from three independent replicates. Data represent mean ± SD, p < 0.05, as calculated by two-sided unpaired Student’s t-test. NI = non-infected and WI = Wnt-independent. f RT-qPCR data showing expression of mature miR-34 relative to U6 from three independent replicates. Data represent mean ± SD, p < 0.05, as calculated by two-sided unpaired Student’s t-test. NI = non-infected and WI = Wnt-independent. g Organoids from miR-34a KO mice do not grow in the absence of Wnt and CHIR99021, whereas organoids from miR-34a/b/c KO mice do grow in the absence of Wnt and CHIR99021. Image representative of two independent replicates. Scale bars: 1 mm. Source data are provided as a Source Data file.