Fig. 3.

PCP enzymes are required for 3D tumorigenesis (Matrigel embedding assays). Cells isolated from the distant metastatic subclones indicated were transfected with non-targeting control siRNAs (siNeg) or siRNAs against the indicated genes (Sigma), trypsinized into single cells, and 1000 cells were embedded in 50 μl of Matrigel discs in triplicate, similar to conventional organoid assays [54] using DMEM with 10% FBS and no additional supplements. Tumoroids were photographed and counted after 3 weeks of tumoroid growth. Knockdown of each individual PCP enzyme impaired tumoroid growth relative to siNeg controls, similar to PGD (see Supplementary Fig. 4 for knockdown efficacies). Statistical significance was calculated using 2-tailed t-tests (*p < 0.05)