Fig 2: YAP is O-GlcNAcylated by OGT at Thr241.(a) YAP O-GlcNAcylation was measured via HRP-labelled Streptavidin (left) and anti-TAMRA antibodies (middle) in Bel-7402 and SMMC-7721 cells. In vitro O-GlcNAcylation of YAP was measured by incubating purified YAP and OGT proteins with UDP-GlcNAc at 37 °C for 90 min before immunoprecipitation with anti-YAP antibodies, followed by western blot using an anti-O-GlcNAc antibody (right). (b) Enzymatic labelling of O-GlcNAc in WT and T241A YAP-FLAG proteins as analysed by anti-TAMRA antibodies and HRP-labelled Streptavidin in Bel-7402 and SMMC-7721 cells. (c) YAP-FLAG was immunoprecipitated by an anti-FLAG antibody in Bel-7402 and SMMC-7721 cells transfected with WT or T241A YAP-FLAG-expressing plasmids. Phosphorylation of YAP was detected using an anti-p-YAP antibody. (d) Mutation of Thr241 induced LATS1-YAP binding under stimulation of O-GlcNAcylation by combined treatment of GlcNAc (4 mM) and PuGNAc (25 μM). Expression plasmids for YAP-FLAG, WT or T241A were transfected into Bel-7402 and SMMC-7721 cells, and cells were treated as indicated for 24 h before analysis. YAP-FLAG was immunoprecipitated with anti-FLAG antibodies, and the association with LATS1 was measured using anti-LATS1 antibodies. (e) Mutation of Thr241 reduced YAP-promoted transformative phenotypes in vitro. Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured with MTT-based assays, soft agar colony formation assays and Caspase 3/7 Glo luciferase assays, respectively, in Bel-7402 and SMMC-7721 cells expressing WT or T241A YAP-FLAG. Scale bar, 500 μm. (f) Mutation of Thr241 diminished YAP-promoted xenograft tumour formation in vivo. The tumour size was measured 72 days after injection of Bel-7402 or SMMC-7721 cells into nude mice. (g) Mutation of Thr241 reduced the half-life of YAP. YAP-FLAG (as indicated) was transfected into Bel-7402 and SMMC-7721 cells, and cells were harvested at the indicated time points after addition of CHX (50 μg ml−1). The expression of YAP-FLAG was normalized to that of GAPDH, and the ‘0 h' point was 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 d–f were analysed by Student's t-test.

Fig 3: O-GlcNAcylation of YAP stimulates tumorigenesis.(a–d) Ectopic expression of YAP rescued the diminished transformative phenotypes caused by OGT knockdown in vitro. The protein expression of OGT, O-GlcNAc, p-YAP and YAP in Bel-7402 and SMMC-7721 cells was measured by WB (a). Cell proliferation, colony formation capacity and Caspase 3/7 activities were measured with an MTT-based assay (b), soft agar colony formation assay (c) and Caspase 3/7 Glo luciferase assay (d), respectively, in Bel-7402 and SMMC-7721 cells under different treatments as indicated. Scale bar, 500 μm. (e,f) Ectopic expression of YAP rescued the xenograft tumour growth inhibited by OGT silencing in vivo. The protein expression of OGT, O-GlcNAc and YAP in xenografts was measured by WB (e). Tumour volumes were monitored for 36 days after subcutaneous injection of Bel-7402 cells into nude mice; n=5 per group (f). Representative IHC images of cleaved Caspase 3 and Ki67 staining in the xenografts formed in nude mice. Scale bar, 500 μm. Representative images from three independent experiments are shown (a–e). **P<0.01 indicates statistical significance. The data from b–d,f were analysed by one-way and two-way ANOVA, respectively.

Fig 4: YAP reversely stimulates O-GlcNAcylation.(a) OGT and YAP increased the intracellular glucose level and glucose uptake in Bel-7402 and SMMC-7721 cells. The intracellular glucose levels were measured directly from cell lysates (1.25 × 106 cells/100 µl of lysis buffer, left). The glucose consumption levels were calculated as the difference between the initial and the remaining glucose levels in the cell culture media of cells subjected to different treatments as indicated (right). (b) OGT, Nudt9 and SLC5A3 were analysed by qPCR or WB in control cells and in Bel-7402 or SMMC-7721 cells with YAP knocked down or overexpressed. (c) Bel-7402 or SMMC-7721 cells were transfected with expression plasmids as indicated, and the mRNA levels of OGT, Nudt9 and SLC5A3 were analysed with qPCR. The promoter activities of OGT, Nudt9 and SLC5A3 genes were measured with a dual luciferase analysis. (d) YAP stimulated O-GlcNAcylation. O-GlcNAcylation was analysed by WB in control cells and in Bel-7402 or SMMC-7721 cells with YAP knocked down (left) or overexpressed (right). (e) Stimulation of O-GlcNAcylation was dependent on YAP. Control and Bel-7402 cells with YAP knocked down were treated with DMSO, PuGNAc (to a final concentration of 25 µM) with GlcNAc (to a final concentration of 4 mM) for 24 h before being harvested to test the indicated proteins using WB (left). The same proteins were also tested by WB in control cells and Bel-7402 cells with YAP knocked down after treatment with the indicated concentration of glucose for 24 h (right). (f) Schematic diagram of a possible mechanism underlying the interaction between O-GlcNAcylation and YAP during tumorigenesis in liver cancer cells. The data represent the means+s.d. from three independent experiments. **P<0.01 indicates statistical significance. The data from a,b (left two panels) and c were analysed by one-way ANOVA, and the data from b (middle two panels) were analysed by Student's t-test.

Fig 5: YAP expression is associated with global O-GlcNAcylation in liver cancer tissues and established liver cancer cell lines.(a–c) TMA of YAP, Ki67, O-GlcNAc, CTGF and TEAD. IHC images of TMA stained with anti-YAP, anti-Ki67, anti-CTGF, anti-TEAD and anti-O-GlcNAc antibody, (a). Enlarged IHC images of YAP and O-GlcNAc staining from the TMA (b). The data were analysed using a chi-square test (c). (d–f) O-GlcNAc and YAP expression in liver cancer and established cell lines. Western blots of O-GlcNAc, TEAD, p-YAP and YAP in liver cancer and adjacent normal liver tissues. The levels of p-YAP were normalized to those of YAP. In addition, the levels of O-GlcNAc and YAP were normalized to those of GAPDH. The levels of the #1 sample of liver cancer were arbitrarily set to 100% (d). Representative images of IHC staining of YAP, TEAD and O-GlcNAc in liver cancer and adjacent normal liver tissues. Scale bar, 500 μM. The liver cancer and normal liver tissue samples are paired (e). YAP and TEAD expression and O-GlcNAc levels in established hepatocyte (THLE-3 and HL-7702) and the indicated liver cancer cell lines detected by IHC and WB, respectively. The levels of YAP and of O-GlcNAc were normalized to those of GAPDH, and the levels of the THLE-3 cells were arbitrarily set to 100% (f). The data are shown as the means+s.d. from three independent experiments (including WB). **P<0.01 indicates statistical significance. Data in c,d,f were analysed using a χ2 test, two-way and one-way ANOVA tests, respectively.