Journal: Cell Death & Disease
Article Title: O-GlcNAcylation of YAP1 promotes lung transplant ischemia-reperfusion injury via binding to HIF1α transcription factor and activating autophagy and mitophagy
doi: 10.1038/s41419-026-08548-w
Figure Lengend Snippet: A Western blot analysis revealed that hypoxia-reoxygenation (0, 2, 4, 6, 12 h) treatment gradually increased the O-GlcNAc modification level of total cellular proteins and the expression of the key modifying enzyme OGT. B Western blot analysis showed that ischemia-reperfusion (2, 6, 12 h) gradually increased the O-GlcNAc modification level of total lung tissue proteins and the expression of OGT. C YAP1 protein immunoprecipitation experiments demonstrated that hypoxia-reoxygenation (0, 2, 4, 6, 12 h) treatment gradually increased the O-GlcNAc modification level of cellular YAP1 protein. D YAP1 protein immunoprecipitation experiments revealed that ischemia-reperfusion (2, 6, 12 h) gradually increased the O-GlcNAc modification level of YAP1 protein in lung tissue. E Flag-YAP1 protein immunoprecipitation with M2 Magnetic Beads experiments revealed that overexpression of wild-type OGT significantly increased the O-GlcNAc modification level of wild-type Flag-YAP1 protein and its binding to the HIF1A transcription factor in alveolar epithelial cells under hypoxia-reoxygenation (12 h). Meanwhile, overexpression of OGT with a serine mutation at the O-GlcNAc modification site had no such effect. Additionally, OGT knockdown significantly inhibited the O-GlcNAc modification level of wild-type Flag-YAP1 protein and its binding to the HIF1A transcription factor in alveolar epithelial cells under hypoxia-reoxygenation (12 h). F Chromatin immunoprecipitation (ChIP) experiments for HIF1α found that overexpression of wild-type OGT significantly increased the binding level of the HIF1A transcription factor to the promoters of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitophagy genes ( FUNDC1 , PINK1 , TBK1 ) in alveolar epithelial cells under hypoxia-reoxygenation (12 h). In contrast, overexpression of OGT with a serine mutation at the O-GlcNAc modification site had no such effect. Moreover, OGT knockdown significantly inhibited the binding level of the HIF1A transcription factor to the promoters of these autophagy and mitophagy genes in alveolar epithelial cells under hypoxia-reoxygenation (12 h). Data represent mean ± SEM ( N = 3), * P < 0.05, ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. G Dual luciferase reporter experiments demonstrated that overexpression of wild-type OGT significantly increased the promoter activity of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitophagy genes ( FUNDC1 , PINK1 , TBK1 ) in alveolar epithelial cells under hypoxia-reoxygenation (12 h). Conversely, overexpression of OGT with a serine mutation at the O-GlcNAc modification site had no such effect. Additionally, OGT knockdown significantly inhibited the promoter activity of these autophagy and mitophagy genes in alveolar epithelial cells under hypoxia-reoxygenation (12 h). Data represent mean ± SEM ( N = 3), * P < 0.05, ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test.
Article Snippet: Afterward, the membrane underwent treatment with 5% skim milk powder at room temperature for 1 h. It was then incubated overnight at 4 °C with primary antibodies against YAP1 (Cat No. 13584-1-AP, 1:1500), p-YAP1 (Cat No. 80694-2-RR, 1:2000), HIF-1α (Cat No. 66730-1-Ig, 1:1000), CYR61 (No. 26689-1-AP, 1:1500), AREG (Cat No. 16036-1-AP, 1:2300), BIRC5 (Cat No. 10508-1-AP, 1:1200), FUNDC1 (Cat No. 28519-1-AP, 1:1500), PINK1 (Cat No. 23274-1-AP, 1:2000), TBK1 (Cat No. 28397-1-AP, 1:1500), HMGB1 (Cat No. 10829-1-AP, 1:1000), DAPK (Cat No. 25136-1-AP, 1:1000), LC3-I/II (Cat No. 14600-1-AP, 1:3000), OGT (Cat No. 11576-2-AP, 1:2000) and O-GlcNAc (Cat No. 65292-1-Ig, 1:3000) (all obtained from Proteintech, Wuhan, China).
Techniques: Western Blot, Modification, Expressing, Immunoprecipitation, Magnetic Beads, Over Expression, Binding Assay, Mutagenesis, Knockdown, Chromatin Immunoprecipitation, Luciferase, Activity Assay
Proteintech is a verified supplier 
