FIG 5.

YAP stability. (A) MT-103A downregulates endogenous YAP. Rat-1 cells stably expressing MT and NIH 3T3 cells and NMuMG cells induced to express EGFP or MT by exposure to doxycycline for 24 h were subjected to SDS-PAGE and Western blotting to detect YAP, MT, and GAPDH. GAPDH served as a loading control. (B) MT-103A does not downregulate YAP at the mRNA level. NIH 3T3 cells were induced to express EGFP (control [Con]) or MT by exposure to doxycycline for 24 h. Total RNA was extracted and subjected to reverse transcription and real-time PCR analysis. (C) MT-103A destabilizes the YAP protein. NIH 3T3 cells were induced to express EGFP or MT by exposure to doxycycline for 48 h, and the last 24 h of induction took place in the presence of the protein synthesis inhibitor cycloheximide. After SDS-PAGE, Western blotting was conducted to detect YAP, MT, and p85. p85 was used as a loading control. The bar graph represents quantification of Western blot data and shows the averages of data from two independent experiments. In the absence of cycloheximide, the difference between WT MT and MT-103A is significant (P < 0.05). In the presence of cycloheximide, the difference between WT MT and MT-103A is more significant (P < 0.001). (D) YAP destabilization is associated with S397 phosphorylation and is proteasome dependent. NIH 3T3 cells were induced to express EGFP or MT by exposure to doxycycline for 24 h, and the proteasome inhibitor bortezomib was added for the last 16 h of induction. After SDS-PAGE, Western blotting was conducted to detect YAP phosphorylation at S397 as well as total YAP, MT, and p85 levels. p85 served as a loading control.