Fig. 4: YAP O-GlcNAcylation regulates its phosphorylation at S397. | Nature Communications

Fig. 4: YAP O-GlcNAcylation regulates its phosphorylation at S397.

From: Protein O-GlcNAcylation coupled to Hippo signaling drives vascular dysfunction in diabetic retinopathy

Fig. 4

A HEK 293T cells transfected with indicated plasmids. pYAP-S397 was analyzed via immunoprecipitation followed by western blot (n = 3 independent experiments). Mean ± SEM, p-values = 0.0068, 0.004 (one-way ANOVA, Dunnett’s test). B Western blot and quantification of pYAP-S397 in HRCECs treated with DMSO or 25 μM PUGNAc for 24 h (n = 3 independent experiments). Mean ± SEM, p = 0.0002 (two-tailed Student’s t-test). C pYAP-S397 in HRCECs treated with DMSO or 15 μM OSMI-1 for 24 h in 25 mM glucose medium (n = 3 independent experiments). Mean ± SEM, p = 0.0053 (two-tailed Student’s t-test). D, E YAP O-GlcNAcylation in HRCECs infected with Ad-Flag-YAP, transfected with siCtrl or siOGT/siOGA under 25 mM glucose (n = 3 independent experiments). Mean ± SEM, p-values: (D) = 0.0091, 0.0318; (E) = 0.0336, 0.0339 (two-tailed Student’s t-test). F, G Relative CTGF and CYR61 mRNA in HRCECs treated as in (D) analyzed by qRT-PCR (n = 3 independent experiments). Mean ± SEM, p-values: (F) = 0.0191, 0.0458; (G) = 0.0298, 0.0443 (two-tailed Student’s t-test). H HRCECs infected with indicated adenovirus, treated with DMSO or 25 μM PUGNAc, and analyzed for pYAP-S397 (n = 3 independent experiments). Mean ± SEM, p = 0.0012 (one-way ANOVA, Dunnett’s test). I Ubiquitination and β-TRCP levels in HRCECs infected with indicated adenovirus and treated with MG132 or vehicle (n = 3 independent experiments). Mean ± SEM, p-values = 0.0011, 0.0002, 0.0196, 0.0125 (one-way ANOVA, Tukey’s test). J YAP stability in HRCECs infected with indicated adenovirus and treated with 50 µg/ml CHX for western blot. Flag-YAP protein levels were quantified as Flag-YAP/β-Actin (n = 3 independent experiments). Mean ± SEM, p-values: 0.0064, <0.0001 (two-way ANOVA, Šídák’s test). K–N Images and quantification of YAP/TAZ (green) within vessels (CD31+ or IsoB4+ areas, red) from OgtWT and OgtiΔEC STZ or OIR mice (n = 4/4 mice in L, 6/6 mice in N). Mean ± SEM, p-values: (L) = 0.0443; (N) = 0.0002 (two-tailed Student’s t-test). *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars: 30 µm in (K, M). Source data provided as a Source Data file.

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