(a) The expression of YAP/TAZ/TEAD target genes involved in cell growth depends on AP-1. MDA-MB-231 cells were transduced with rtTA and doxycycline-inducible JUN-DN. Cells were left untreated (CO) or treated with 1 μg/ml doxycycline for 48h. As control, JUN-DN reduces the expression of FOSL1, CTGF and ANKRD1 (see Supplementary Fig. 6a). All expression levels are normalized to GAPDH.
(b) Control and TAZS89A-overexpressing MII cells were transfected with the indicated siRNAs and tested for mammosphere formation. Data are mean+SD of n=6 biological replicates from a representative experiment. See Supplementary Figure 6c for a comparison with control MII cells.
(c) Quantification and representative pictures of colonies formed by the indicated MCF10A derivatives in soft agar assays. Only background/not growing cell clusters were formed by control and MCF10A+AP-1 cells, and were not counted as colonies. Data are presented as mean+SD of n=3 biological replicates from a representative experiment. Magnification is the same for all pictures.
(d) Quantification and representative pictures of primary mammospheres formed by the indicated MCF10A derivatives. Data are presented as mean+SD of n=6 biological replicates from a representative experiment. Magnification is the same for all pictures.
(e-g) YAP and AP-1 synergize to promote tumor growth. (e) Representative IHC pictures of xenografts formed by the indicated cell lines. MCF10A cells were stained with a human-specific pan-cytokeratin antibody. (f) Tumor volumes at harvesting (individual tumors are plotted, line is the mean). (g) Quantification of Ki67-positive cells in tumor sections; data are mean + SEM of at least n=8 different samples.
(h) YAP5SA and AP-1 cooperate to activate YAP/TAZ/TEAD proliferative program in MCF10A cells. MCF10A derivatives were grown on a thick Matrigel coating for one week before harvesting for RNA extraction. mRNA levels of the indicated genes were evaluated by qRT-PCR and normalized to GAPDH.
See Methods for reproducibility of experiments.