Fig 2: Downregulation of CRB3 confers CSC traits on breast cancer cells through the TAZ/ß-catenin cascade. (a) Western blot detecting the expressions of AMOT, TAZ and ß-catenin. (b) CoIP/western blot analysis showing endogenous AMOT bound to TAZ. (c) Fluorescence-activated cell sorting of CD44high/CD24low population. (d) Western blot of TAZ and stem cell markers. (e, g) Representative images and quantification of formed mammospheres. (f) Western blot of ß-catenin and stem cell markers. XAV939 was used to inhibit ß-catenin. (h) Flow cytometry analysis of the stem cell marker ALDH and the CD44high/CD24low population. All data are presented as mean±s.e.m. and statistical significance was calculated using a two-tailed t-test.

Fig 3: CRB3 downregulation induces activation of TAZ and ß-catenin. (a) Western blot of CRB3 in human mammary epithelial cells. (b) Western blot of the Hippo pathway components. (c) Western blot of ß-catenin, ß-TrCP and ßTrcp substrates Smad4 after CRB3 knockdown. (d) Cytoplasmic and nuclear expression of TAZ and ß-catenin. (e) MCF 10A cells were transfected with the TEAD-luciferase reporter and TEAD promoter activity was detected by luciferase assay. (f) Real-time PCR showed that CRB3 knockdown increased expressions of TAZ target gene CYR61 and CTGF. (g) Luciferase assay of TOPFLASH or control FOPFLASH as a measure of ß-catenin/TCF activity. (h) Real-time PCR of TERT. (i) Localization of TAZ, ß-catenin and YAP as shown by IF. All data are presented as mean±s.e.m. and statistical significance was calculated using a two-tailed t-test.