Figure 4

YAP/TAZ Activity Dictates Caveolar Protein Levels

(A) Dot plots of in silico bioinformatics analysis of data obtained from cancer cell line encyclopedia (CCLE) in cell lines from 967 subjects. The analysis revealed strong positive correlation between established YAP/TAZ target genes (CYR61 and CTGF) as well as between CYR61 and CAV1 or CAVIN1. However, there was no correlation for HPRT1 or for another endolysosomal gene, such as FLOTILLIN2 (FLO2), serving as negative controls (Figure S5A). R values were calculated for each correlation; p < 0.05; Pearson’s correlation coefficient.

(B) Western blots from YAP KO U2OS cells with stable shRNA-induced knockdown of TAZ or control (Figure S5D).

(C) Western blots from TAZ KO U2OS cells with stable knockdown of YAP or control (Figure S5D).

(D) Western blots from NF2 KO and WT U2OS cells (Figure S5B).

(E and F) Confocal image of U2OS WT (E) and NF2 KO cells labeled for YAP/TAZ (green) and DAPI (blue; F). Scale bars represent 20 μm.

(G) Dot blots of nuclear-to-cytoplasmic ratio of YAP/TAZ, quantified of images as in (E) and (F). Each dot represents one cell. Means ± SEM.

(H) Cell lysates from WT, NF2 KO cells exogenously re-expressing NF2, or NF2 KO control cells were separated in a PhosTag gel and probed for YAP.

(I) Lysates, as in (H), separated on a conventional SDS-gel and processed for western blotting.

(J) qPCR data from NF2 re-expressors, relative to NF2 KO U2OS control cells. NF2 re-expression reduces expression of CYR61, CAV1, and CAVIN1. Means ± SD.

(K) qPCR data from NF2 KO U2OS shTEADs relative to control cells. TEAD knockdown reduces expression of CYR61, CAV1, and CAVIN1. Means ± SD (Figures S5B and S5C).

(L) Micrographs from epidermis cells from WT zebrafish embryos. Arrows: examples of caveolae at the plasma membrane.

(M) Micrographs from epidermis cells from mutant (yap^−/−:taz^−/−) zebrafish embryos show no caveolae. Scale bars in (L) and (M) represent 500 nm.

(N) Quantification of caveolae from micrographs; each dot represents data from one fish embryo. Means ± SEM.

(O) qPCR analysis of zebrafish from 5 embryos of each genotype. Means ± SD.