Fig. 3.

YAP and TEAD are required for RHAMM expression, ERK activity, and BCCMI, whereas ectopic expression of RHAMM bypasses the requirement. (A and B) Knockdown of YAP or TEAD1/3/4 decreased RHAMM transcription (A) and protein (B) levels. MDA-MB-231 cells were infected with the indicated shRNA lentiviruses, and RHAMM mRNA (A) and protein (B) levels were determined by real-time RT-PCR and Western blot, respectively. (B) (Right) Quantification of the protein bands. (C) Knockdown of YAP or TEAD1/3/4 reduced CTGF mRNA levels (experiments as in A). (D and E) Overexpression of YAP increased RHAMM mRNA (D) and protein (E) levels. HEK 293T cells were transiently transfected with the indicated YAP plasmids, and, 48 h later, total RNA was extracted to detect RHAMM mRNA by using real-time RT-PCR (D). (E) Cell lysates were immunoblotted with the indicated antibodies (Left), and the protein bands were quantified (Right). (F) Knockdown of YAP or TEAD1/3/4 decreased ERK phosphorylation levels. Experiments were as in B except Western blot was performed with the indicated antibodies. (G and H) Knockdown of YAP or TEAD1/3/4 decreased MDA-MB-231 cell migration (G, Left) and invasion (H, Left), whereas reexpression of RHAMM rescued the effects (G and H, Right). Data are shown as mean ± SD of three independent experiments. Student two-tailed t test was used for statistical analysis (*P < 0.05 and ***P < 0.001).