Figure 2.

JOSD2 plays vital role in CCA proliferation and stabilizes YAP/TAZ through deubiquitinase activity. The stably silence of JOSD2 remarkably inhibits CCA proliferation (A) and colony formation (B). The results represent the mean ± SD of three independent experiments; ∗∗P < 0.01, ∗∗∗P < 0.001. (C) Knockdown of JOSD2 down-regulates YAP and TAZ protein levels. (D) Over-expression of JOSD2-WT but not its catalytically inactive mutant JOSD2-C24A greatly increases YAP/TAZ protein levels. (E) Knockdown of JOSD2 dramatically decreases YAP/TAZ protein stability. The protein levels were quantified by Image J. (F) Silencing of JOSD2 in 293T cells distinctly reduces fluorescence signals in 8 × GTIIC-luciferase system (shJOSD2#1 inhibition ratio = 78.2%, shJOSD2#2 inhibition ratio = 79.5%) and WWTR-luciferase reporter system (shJOSD2#1 inhibition ratio = 70.3%, shJOSD2#2 inhibition ratio = 77.6%). The results represent the mean ± SD of three independent experiments; ∗∗∗P < 0.001. (G) Representative images of immunofluorescence with YAP in green and DAPI in blue shows nuclear translocation of YAP in JOSD2 over-expressed HuCCT-1 cells. The nuclear vs cytoplasm ratio was determined in 50 cells per cohort by Image J and represented as the mean ± SEM; ∗P < 0.05. (H) Down-regulation of YAP/TAZ caused by JOSD2 depletion in CCLP-1 cells can be rescued by MG132 (10 μmol/L, 6 h). (I) JOSD2 can antagonize SCF^β−TRCP E3 Ligase to stabilize YAP/TAZ protein levels in RBE cells.