dapk  (Proteintech)

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, B Immunofluorescence confocal laser scanning microscopy detected a gradual increase in mitochondrial autophagy levels in cells subjected to hypoxia-reoxygenation (0, 2, 4, 6, 12 h). Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by one-way ANOVA along with Tukey’s post hoc test. C Western Blot analysis showed a gradual increase in the expression level of the autophagy protein LC3-II in cells subjected to hypoxia-reoxygenation (0, 2, 4, 6, 12 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by one-way ANOVA along with Tukey’s post hoc test. D Realtime PCR revealed a gradual increase in the expression levels of autophagy genes ( HMGB1 , DAPK ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to hypoxia-reoxygenation (0, 2, 4, 6, 12 h). Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. E Western Blot analysis showed a gradual increase in the protein expression levels of autophagy genes ( HMGB1 , DAPK ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to hypoxia-reoxygenation (0, 2, 4, 6, 12 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. F Transmission electron microscopy observations revealed a gradual increase in the morphological phenotypes of mitochondrial autophagy in cells subjected to hypoxia-reoxygenation (0, 2, 4, 6, 12 h).

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: Immunofluorescence, Confocal Laser Scanning Microscopy, Western Blot, Expressing, Transmission Assay, Electron Microscopy

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 HE staining experiments assessed gradually increasing pathological damage and lung edema in lung tissue at different time points of ischemia-reperfusion (2, 6, 12 h). Data represent mean ± SEM ( N = 3), * P < 0.05, ** P < 0.01, *** P < 0.001, compared with indicated group by one-way ANOVA along with Tukey’s post hoc test. B Realtime PCR revealed a gradual increase in the expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to ischemia-reperfusion (2, 6, 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. C Western Blot analysis showed a gradual increase in the protein expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to ischemia-reperfusion (2, 6, 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. D Realtime PCR revealed a gradual increase in the expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to ischemia-reperfusion (2, 6, 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. E Western Blot analysis showed a gradual increase in the protein expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to ischemia-reperfusion (2, 6, 12 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. F TUNEL assays demonstrated a gradual increase in apoptosis levels in lung epithelial cells subjected to ischemia-reperfusion (2, 6, 12 h). Data represent mean ± SEM ( N = 3), ** 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: Staining, Expressing, Western Blot, TUNEL Assay

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 Realtime PCR revealed that YAP1 knockdown significantly inhibited the increased expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to hypoxia-reoxygenation (12 h). Data represent mean ± SEM ( N = 3), * P < 0.05, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. B Western Blot analysis showed that YAP1 knockdown significantly inhibited the increased protein expression levels of Hippo-YAP signaling pathway molecules (YAP1, CYR61, AREG, and BIRC5) in cells subjected to hypoxia-reoxygenation (12 h). Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. C Immunofluorescence confocal laser scanning microscopy detected that YAP1 knockdown with two independent shRNAs targeting YAP1 (shYAP1-1 and shYAP1-2) significantly inhibited the increased mitochondrial autophagy levels in cells subjected to hypoxia-reoxygenation (12 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. D Realtime PCR revealed that YAP1 knockdown significantly inhibited the increased expression levels of autophagy genes (HMGB1, DAPK ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to hypoxia-reoxygenation (12 h). Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. E Western Blot analysis showed that YAP1 knockdown significantly inhibited the increased protein expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to hypoxia-reoxygenation (12 h). F Transmission electron microscopy observations revealed that YAP1 knockdown significantly inhibited the morphological phenotypes of mitochondrial autophagy induced by hypoxia-reoxygenation (12 h) in cells. Data represent mean ± SEM ( N = 3), *** 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: Knockdown, Expressing, Western Blot, Immunofluorescence, Confocal Laser Scanning Microscopy, Transmission Assay, Electron Microscopy

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 HE staining experiments assessed that YAP1 knockdown significantly inhibited the increased pathological damage and lung edema in lung tissue subjected to ischemia-reperfusion (6 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by one-way ANOVA along with Tukey’s post hoc test. B Realtime PCR revealed that YAP1 knockdown significantly inhibited the increased expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to ischemia-reperfusion (6 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. C Western Blot analysis showed that YAP1 knockdown with two independent shRNAs targeting YAP1 (shYAP1-1 and shYAP1-2) significantly inhibited the increased protein expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to ischemia-reperfusion (6 h). Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. D Realtime PCR revealed that YAP1 knockdown significantly inhibited the increased expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to ischemia-reperfusion (6 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. E Western Blot analysis showed that YAP1 knockdown with two independent shRNAs targeting YAP1 (shYAP1-1 and shYAP1-2) significantly inhibited the increased protein expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitochondrial autophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to ischemia-reperfusion (6 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. F TUNEL assays demonstrated that YAP1 knockdown with two independent shRNAs targeting YAP1 (shYAP1-1 and shYAP1-2) significantly inhibited the increased apoptosis levels in lung epithelial cells subjected to ischemia-reperfusion (6 h). Data represent mean ± SEM ( N = 3), ** P < 0.01, *** P < 0.001, compared with indicated group by one-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: Staining, Knockdown, Expressing, Western Blot, TUNEL Assay

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

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 Real-time qPCR analysis revealed that OGT knockdown significantly inhibited the increased expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells treated with 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. B Western blot analysis showed that OGT knockdown significantly inhibited the increased protein expression levels of Hippo-YAP signaling pathway molecules (YAP1, CYR61, AREG, and BIRC5) in cells treated with 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. C Immunofluorescence confocal laser scanning microscopy experiments demonstrated that OGT knockdown significantly inhibited the increased level of mitophagy in cells treated with 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. D Real-time qPCR analysis found that OGT knockdown significantly inhibited the increased expression levels of autophagy genes ( HMGB1 , DAPK ) and mitophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells treated with 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. E Western blot analysis revealed that OGT knockdown significantly inhibited the increased protein expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells treated with 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. F Transmission electron microscopy observations showed that OGT knockdown significantly inhibited the morphological phenotypes of mitophagy induced by hypoxia-reoxygenation (12 h) treatment in cells.

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: Knockdown, Expressing, Western Blot, Immunofluorescence, Confocal Laser Scanning Microscopy, Transmission Assay, Electron Microscopy

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 HE staining experiments assessed that OGT knockdown significantly inhibited the increased pathological damage and lung edema in lung tissue subjected to ischemia-reperfusion (6 h) during lung transplantation. Data represent mean ± SEM ( N = 3), * P < 0.05, ** P < 0.01, *** P < 0.001, compared with indicated group by one-way ANOVA along with Tukey’s post hoc test. B Real-time qPCR analysis found that OGT knockdown significantly inhibited the increased expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to ischemia-reperfusion (6 h) during lung transplantation. Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. C Western blot analysis showed that OGT knockdown significantly inhibited the increased protein expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) in cells subjected to ischemia-reperfusion (6 h) during lung transplantation. Data represent mean ± SEM ( N = 3), * P < 0.05, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. D Real-time qPCR analysis revealed that OGT knockdown significantly inhibited the increased expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to ischemia-reperfusion (6 h) during lung transplantation. Data represent mean ± SEM ( N = 3), * P < 0.05, *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. E Western blot analysis demonstrated that OGT knockdown significantly inhibited the increased protein expression levels of autophagy genes ( HMGB1 , DAPK , and LC3-II ) and mitophagy genes ( FUNDC1 , PINK1 , TBK1 ) in cells subjected to ischemia-reperfusion (6 h) during lung transplantation. Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by two-way ANOVA along with Tukey’s post hoc test. F TUNEL experiments found that OGT knockdown significantly inhibited the increased level of apoptosis in lung epithelial cells subjected to ischemia-reperfusion (6 h) during lung transplantation. Data represent mean ± SEM ( N = 3), *** P < 0.001, compared with indicated group by one-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: Staining, Knockdown, Transplantation Assay, Expressing, Western Blot, TUNEL Assay