Journal: Nature Communications

Article Title: The essential role of YAP O-GlcNAcylation in high-glucose-stimulated liver tumorigenesis

doi: 10.1038/ncomms15280

Figure Lengend Snippet: ( 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.

Article Snippet: The lentiviral-based OGT expression plasmid and OGT-shRNA (sh4) were purchased from Origene (Beijing, China), and the OGT-sh7 was purchased from Genechem (Shanghai, China) The expression plasmids encoding YAP-sh1&2, YAP, YAP-FLAG, TEAD4-Myc, CREB-HA, βTrCP-FLAG, c-Fos and the pUAS-Luc/TEAD-Gal4 system were constructed, as previously described by us .

Techniques: Infection, shRNA, Western Blot, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation