Figure 5. Treatment with BET inhibitor blunts YAP/TAZ-addicted breast tumors.

a) Heat map showing the regulation of YAP/TAZ target genes in triple negative breast cancer cells after YAP/TAZ depletion (siYT1, siYT2) or treatment with BET inhibitors (1µM, 24h). Expression values are normalized to cells transfected with control (siRNA) and to GAPDH.

b) Left: viability curves of TNBC cells treated with increasing doses of JQ1 (1nM to 100µM). Data are mean of n=8 independent wells (independently treated and evaluated). Right: IC50 of listed cell lines.

c) Proliferation of BT20 cells is not impaired by YAP/TAZ depletion, whereas all cells sensitive to JQ1 are also affected by YAP/TAZ depletion.

d) BRD4 downregulation by shRNAs impairs colony formation by YAP5SA-overexpressing MCF10A cells in soft agar. Data are presented as individual data points (n=3 independent samples) + average (bar), from one of three experiments providing similar results. Similar results were obtained in MDA-MB-231 cells, whose colony-forming capacity depends on endogenous YAP/TAZ (see Supplementary Fig. 5b).

e) Quantification of colonies formed by YAP5SA-overexpressing MCF10A cells in soft agar, upon treatment with 0,1µM or 1µM JQ1 for the entire experiment. Data are presented as in d. Similar results were obtained in MDA-MB-231 cells (see Supplementary Fig. 5c).

f) Inhibition of the growth of colonies initiated by YAP5SA-overexpressing MCF10A cells in soft agar upon addition of JQ1 (1µM) to culture medium 8 or 15 days after seeding (treatment with JQ1 at day1 is presented as reference for maximal inhibition). Data are presented as in d.

g) Representative hematoxylin and eosin (H&E) staining of sections of mammary glands from MMTV-Cre;Apc^+/+, MMTV-Cre;Apc^fl/fl, or MMTV-Cre;Apc^fl/fl;Yap^fl/fl;Taz^fl/fl mice. Scale bar is 0.1 mm. The same phenotype was observed in at least 4 mice per each experimental group.

h) Representative immunofluorescence (IF) pictures of mammary glands from the indicated mice, showing YAP accumulation in the nuclei of epithelial cells, expansion of luminal cells (K8-positive) and discontinuities in the basal layer of K14-positive cells in MMTV-Cre;Apc^fl/fl. Ducts of MMTV-Cre;Apc^fl/fl;Yap^fl/fl;Taz^fl/fl mice display a normal morphology. Scale bar is 25 µm. IF was performed on sections derived from 4 mice per each genotype.

i) Representative H&E staining of sections of mammary glands from MMTV-Cre;Apc^fl/fl mice, treated with vehicle (n=5) or BAY-BET inhibitor (n=5) for 6 weeks. All scale bars are 0.1mm. See BAY-BET-inhibitor has no effect on the histological appearance of mammary glands of Apc^fl/fl (Cre-negative) littermates (see Supplementary Fig. 5g).

j) Representative IF pictures of mammary glands from MMTV-Cre;Apc^fl/fl mice, treated with vehicle (n=5) or BAY-BET inhibitor (n=5) for 6 weeks, showing that treatment with BET inhibitor restores normal distribution of the luminal marker K8 and the basal marker K14 in the mammary ducts. Scale bars are 25 µm. See Supplementary Fig. 5h for normal K8/K14 staining in Apc^fl/fl (Cre-negative) mice treated with BAY-BET-inhibitor.