Figure 3: O-GlcNAcylation enhances YAP stability by inhibiting ßTrCP.

(a,b) O-GlcNAcylation increased YAP stability. Protein synthesis was blocked by treatment with CHX (50 μg ml⁻¹) for the indicated times. The half-life of endogenous YAP in Bel-7402 and SMMC-7721 cells treated with DMSO or PuGNAc (25 μM) with or without GlcNAc (4 mM) for 24 h (a) or in Bel-7402 and SMMC-7721 cells infected with the indicated OGT shRNA (b), as measured by western blot analysis. The levels of YAP were normalized to those of GAPDH, and the 0 h points were arbitrarily set to 100%. (c) OGT reversed βTrCP-induced YAP degradation. YAP expression was examined in Bel-7402 and SMMC-7721 cells with βTrCP overexpressed in the presence or absence of transfection with an increasing amount of OGT-Myc plasmid (transfected with 1–3 μg of OGT-Myc-expressing plasmids). The levels of YAP were normalized to those of GAPDH, and the data from the untreated group (lane 1) were arbitrarily set to 100%. (d) Stimulation of O-GlcNAcylation blocked the interaction between βTrCP and YAP. Bel-7402 and SMMC-7721 cells were treated with DMSO or PuGNAc with or without GlcNAc for 24 h. YAP was immunoprecipitated by anti-YAP antibodies, and the association with βTrCP was detected by anti-βTrCP antibodies. The levels of βTrCP were normalized to those of YAP in the IP samples, and the data from cells treated with DMSO were arbitrarily set to 100%. (e) OGT inhibited interactions between βTrCP and YAP. Bel-7402 and SMMC-7721 cells were infected with the indicated OGT shRNA. YAP was immunoprecipitated by anti-YAP antibodies, and the association with βTrCP was detected by anti-βTrCP antibodies. The levels of βTrCP were normalized to those of YAP in the IP samples, and the data from GFP-sh were arbitrarily set to 100%. Representative images are shown, and the data are expressed as the means+s.d. from three independent experiments. **P<0.01 indicates statistical significance. The data from c–e were analysed by one-way ANOVA.