Journal: Allergy

Article Title: Staphylococcus aureus causes aberrant epidermal lipid composition and skin barrier dysfunction

doi: 10.1111/all.15640

Figure Lengend Snippet: Expression of ELOVLs in human primary keratinocytes and organotypic skin cultures stimulated with Staphylococci . Gene expression of ELOVLs in organotypic skin cultures stimulated with S . epidermidis (A), MSSA (B), and MRSA (C). Data are representative of three independent experimental repetitions using three different lots of HEKs S . epidermidis , MSSA, and MRSA, respectively. n = 9 per group. Immunostaining intensities of ELOVL3 and ELOVL4 in organotypic skin cultures stimulated with MRSA (D). Arrows point to ELOVL3 and ELOVL4 staining (red). Wheat germ agglutinin-conjugated FITC (green) was used to stain the cytoskeleton. DAPI stained nuclei (blue). n = 3. Scale bar = 50 μm. All data are shown as the median and interquartile range. * p < .05, ** p < .01, *** p < .001 by the Kruskal–Wallis test with the post hoc test. ELOVL, elongation of very long chain fatty acids protein; MFI, mean fluorescence intensity; MRSA, methicillin-resistant Staphylococcus aureus ; MSSA, methicillin-sensitive Staphylococcus aureus.

Article Snippet: Then, cells were stimulated with three strains of live S . epidermidis (ATCC ® -12228 TM , ATCC ® -49461 TM , and ATCC ® -700296 TM , Manassas, VA, 0.001 or 0.01 CFU/well), three strains of methicillin-sensitive S . aureus (MSSA) (ATCC ® -29213 TM , ATCC ® -49230 TM , and SMC1801–45, Asian Bacterial Bank, Seoul Korea, 0.001 or 0.01 CFU/well), or three strains of MRSA (ATCC ® -BAA-1556 TM , ATCC-700699 TM , and SMC1803–07, Asian Bacterial Bank, 0.001 or 0.01 CFU/well) for an additional 24 h, prior to RNA collection.

Techniques: Expressing, Gene Expression, Immunostaining, Staining, Fluorescence

Journal: Allergy

Article Title: Staphylococcus aureus causes aberrant epidermal lipid composition and skin barrier dysfunction

doi: 10.1111/all.15640

Figure Lengend Snippet: Expression of keratinocyte-derived cytokines in human primary keratinocytes stimulated with Staphylococci . HEKs were Ca2+ differentiated and stimulated with three different strains of S . epidermidis , MSSA or MRSA for 8 or 20 h. Gene expression of keratinocyte-derived cytokines in HEKs stimulated with MSSA (A) and MRSA (B) was evaluated by RT-PCR. n = 27 per group. Protein levels of TNF-α (C) and IL-1β (D) in HEKs culture media were measured by ELISA. n = 4 per group. Data are representative of three independent experimental repetitions using three different lots of HEKs. All data are shown as the median and interquartile range. * p < .05, ** p < .01, *** p < .001, **** p < .0001 by the Kruskal–Wallis test with the post hoc test. MRSA, methicillin-resistant Staphylococcus aureus ; MSSA, methicillin-sensitive Staphylococcus aureus ; RT-PCR, reverse transcription-polymerase chain reaction.

Article Snippet: Then, cells were stimulated with three strains of live S . epidermidis (ATCC ® -12228 TM , ATCC ® -49461 TM , and ATCC ® -700296 TM , Manassas, VA, 0.001 or 0.01 CFU/well), three strains of methicillin-sensitive S . aureus (MSSA) (ATCC ® -29213 TM , ATCC ® -49230 TM , and SMC1801–45, Asian Bacterial Bank, Seoul Korea, 0.001 or 0.01 CFU/well), or three strains of MRSA (ATCC ® -BAA-1556 TM , ATCC-700699 TM , and SMC1803–07, Asian Bacterial Bank, 0.001 or 0.01 CFU/well) for an additional 24 h, prior to RNA collection.

Techniques: Expressing, Derivative Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction

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 a gradual increase in the expression levels of Hippo-YAP signaling pathway molecules (CTGF, CYR61, AREG, and BIRC5) 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. B Western Blot analysis showed a gradual increase in the protein expression levels of Hippo-YAP signaling pathway molecules (YAP1, phosphorylated YAP1, CYR61, AREG, and BIRC5) in cells subjected to hypoxia-reoxygenation (0, 2, 4, 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, D TUNEL assays demonstrated a gradual increase in apoptosis levels in lung epithelial cells subjected to hypoxia-reoxygenation (0, 2, 4, 6, 12 h). Data represent mean ± SEM ( N = 3), * P < 0.05, *** P < 0.001, compared with indicated group by one-way ANOVA along with Tukey’s post hoc test. E ELISA experiments showed a gradual increase in the secretion of inflammatory cytokines (IL-10, MCP1, and ICAM1) 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.

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: Expressing, Western Blot, TUNEL Assay, Enzyme-linked Immunosorbent 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 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 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

Journal: PLoS ONE

Article Title: Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

doi: 10.1371/journal.pone.0053110

Figure Lengend Snippet: The reported lumbrokinase amino acid sequences were from GenBank. Identical residues are shadowed with the same color.

Article Snippet: The standard lumbrokinase product (1200 U/mg) was purchased from Baiao Pharmaceuticals Co., LTD (Beijing, China).

Techniques:

Journal: PLoS ONE

Article Title: Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

doi: 10.1371/journal.pone.0053110

Figure Lengend Snippet: The purified refolded rCST1 (A) and standard lumbrokinase (1200 u/mg) (B). Tube no. 1: negative control 1×PBS; Tube no 2 to 6: sample with concentrations of 5, 10, 20, 40 and 80 mg/mL.

Article Snippet: The standard lumbrokinase product (1200 U/mg) was purchased from Baiao Pharmaceuticals Co., LTD (Beijing, China).

Techniques: Purification, Negative Control

Journal: PLoS ONE

Article Title: Cloning, Expression and Characterization of a Gene from Earthworm Eisenia fetida Encoding a Blood-Clot Dissolving Protein

doi: 10.1371/journal.pone.0053110

Figure Lengend Snippet: Purified refolded rCST1 and standard lumbrokinase (1200 u/mg) clot lysis was monitored by incubating a tube of clotted blood with different concentrations (0, 5, 10, 20, 40 and 80 mg/mL) of lumbrokinase at 37°C for 2 h. Weight difference between untreated and treated samples as expressed as % of clot lysis. The data shown are means of three replicates. Values are means ± SD (n = 3). Regressive equation of standard lumbrokinase: Y = 0.008x-0.3068; correlation coefficient R 2 = 0.97482.

Article Snippet: The standard lumbrokinase product (1200 U/mg) was purchased from Baiao Pharmaceuticals Co., LTD (Beijing, China).

Techniques: Purification, Lysis

Journal: Cell reports

Article Title: Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade

doi: 10.1016/j.celrep.2021.110021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: VECTASHIELD Antifade Mounting Medium with DAPI , Vector Labs , H-1200-10.

Techniques: Recombinant, Cell Stimulation, Protease Inhibitor, Plasmid Preparation, Transfection, Staining, Cell Isolation, Isolation, Enzyme-linked Immunospot, Labeling, AST Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A ) HeLa cells were cultured under the indicated oxygen conditions for 24 hours and subjected to DNA microarray analysis. Of 34 genes that exhibited more than 10-fold induction upon hypoxia, the top 4 genes harboring the N-terminus signal peptide are listed. ( B and C ) HeLa cells were cultured under the indicated oxygen conditions for the indicated periods, and subjected to qPCR for the indicated genes. ( D ) After being cultured under the indicated oxygen conditions for 48 hours, cell lysates were subjected to qPCR. ( E ) Changes in the SPINK1 mRNA levels in HeLa cells were quantified at the indicated time points during (prehypoxia) and after (reoxygenation) the severe hypoxic treatment and represented as mean ± SD. ( F ) After the same treatment as in D , culture media were subjected to the ELISA assay. ( G ) Scatter plot for correlation analysis between SPINK1 mRNA levels and secreted SPINK1 protein levels in cells cultured under the indicated oxygen conditions for the indicated periods. ( H – K ) The indicated cells were transfected with either pcDNA4/SPINK1 (SPINK1) or its EV and cultured for 48 hours. Then, both culture media and cell lysates were subjected to Western blotting using the indicated antibodies ( H and J ), and then, cells were irradiated with the indicated doses of γ-rays and subjected to the clonogenic survival assay ( I and K ). The exogenously expressed SPINK1 was detected using anti-myc tag Ab ( H and J ). Data are represented as mean ± SD ( B , D , F , I , and K ; n = 3 in B – G , n = 6 in I and K ). Two-tailed Student’s t test. * P < 0.05, *** P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Cell Culture, Microarray, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Irradiation, Clonogenic Cell Survival Assay, Two Tailed Test, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A – D ) HeLa cells were cultured under the indicated oxygen conditions in the presence or absence of 5 μg/mL Act D for 24 hours ( A ), 100 μM deferoxamine for 48 hours ( B ), 2 mM DMOG for 24 hours ( C ), or 3 μM MG132 for 12 hours ( D ) and subjected to qPCR. ( E – G ) HeLa cells were transfected with the indicated siRNA or scramble siRNA for negative control, cultured under the indicated oxygen conditions for 24 hours, and subjected to qPCR. ( H and I ) After simultaneously silencing HIF-1α, HIF-2α, and HIF-3α using 2 kinds of mixtures using HIF-αs and siRNAs (mixture-1 and mixture-2), HeLa cells were cultured under the indicated oxygen conditions for 24 hours and subjected to qPCR ( H ) or the ELISA assay ( I ). ( J and K ) The same experiments as in H and I were conducted after silencing HIF-1β. Scramble siRNA was used as a negative control. Data are represented as mean ± SD ( n = 3). Two-tailed Student’s t test ( A – D ). One-way ANOVA with Dunnett’s test ( E – K ). * P < 0.05, ** P < 0.01, *** P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; Act D, actinomycin D; DMOG, dimethyloxallyl glycine.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Cell Culture, Transfection, Negative Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A ) HeLa tumor xenografts were stained with antibodies against a hypoxia marker, pimonidazole (green), or SPINK1 (red). Blue, counter staining with Hoechst 33342. The dotted line represents the outside edge of the pimonidazole-positive regions. Scale bar: 50 μm. ( B – E ) After 24 hours serum starvation, DU145 cells were precultured in the presence or absence of 100 ng/mL rSPINK1 for 24 hours, treated with the indicated dose of γ-ray irradiation, and subjected to the clonogenic survival assay ( B ), colorimetric cell viability assay ( C ), and FACS analysis for the cell cycle status ( D ) and sub-G 1 fraction ( E ). The cells were precultured and irradiated under the indicated oxygen conditions in C . ( F and G ) After transfection with either pcDNA4/SPINK1 (SPINK1) or its EV, the indicated cells were precultured under mild hypoxic conditions (O 2 = 3%) for 48 hours, treated with the indicated dose of γ-ray irradiation under the same oxygen conditions as the preculture, and subjected to the clonogenic survival assay. ( H – K ) The indicated cells were transfected with either pcDNA4/SPINK1-ΔSP (SPINK1-ΔSP) or its EV and cultured for 48 hours. Then, both the culture media and cell lysates were subjected to Western blotting using the indicated antibodies ( H and J ), and then, cells were irradiated with the indicated doses of γ-rays and subjected to the clonogenic survival assay ( I and K ). The exogenously expressed SPINK1-ΔSP was detected using anti-myc tag Ab ( H and J ). ( L ) After 24 hours serum starvation, DU145 cells were treated with or without 100 ng/mL rSPINK1 in combination with SPINK1-neutralizing antibody or control IgG (0.5 μg/mL) for 24 hours and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD ( n = 3 in C – E and L , and n = 6 in B , F , G , I , and K ). Student’s t test. * P < 0.05, ** P < 0.01. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Staining, Marker, Irradiation, Clonogenic Cell Survival Assay, Viability Assay, Transfection, Cell Culture, Western Blot, Control, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A – C ) DU145/EV and DU145/SPINK1 cells were cultured under the indicated oxygen conditions for 48 hours and subjected to qPCR ( A ) and the ELISA assay ( B ) or treated with the indicated dose of γ-ray irradiation for the clonogenic survival assay ( C ). ( D and E ) DU145/EV or SPINK1 xenografts were locally irradiated at a dose of 0 (solid lines) or 10 (dotted lines) Gy. When the volumes of the xenografts reached the same sizes as those on day 0, plasma SPINK1 levels were quantified by ELISA assays ( D ). Tumor growth was analyzed after the treatment ( E ). Data are represented as mean ± SD ( n = 3 in A and B , n = 6 in C , n = 5 in D , and n = 9–10 in E ). Two-tailed Student’s t test. *** P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Clonogenic Cell Survival Assay, Clinical Proteomics, Two Tailed Test, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A – D ) Four days after being transfected with either pcDNA4/SPINK1 or its EV, DU145/EGFP-53BP1-M ( A and B ) and DU145 ( C and D ) cells were irradiated with 0 or 4 Gy of γ-rays in the presence or absence of EGFR-I III ( D ), and the DNA DSBs detected as EGFP-53BP1 foci ( A and B ) or as γH2AX foci ( C and D ) were analyzed 2 hours ( A and B ) or 15 minutes ( C and D ) after the radiation. ( A ) Immunocytochemical analysis. Green, EGFP-53BP1 foci; blue, counter staining using Hoechst 33342. Scale bar: 10 μm. ( B – D ) The number of foci increased by 4 Gy γ-IR was calculated by subtracting the number of foci at 0 Gy from that at 4 Gy under each condition and represented as dot plots with mean ± SD. ( E and F ) DU145 cells were irradiated with 0 or 4 Gy of γ-ray in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 0.5 μM EGFR-I III ( E ), or with control IgG or 10 μg/mL cetuximab ( F ), and subjected to colorimetric cell viability assays. ( G ) The same experiment as in was conducted in the presence or absence of EGFR-I III. Data are represented as mean ( n > 1000 in B – D ) and mean ± SD ( n = 3 in E and F , and n = 6 in G ). Two-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector. EV, empty vector; EGFR-I III, EGFR Inhibitor III; DSBs, double-strand breaks.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Transfection, Irradiation, Staining, Control, Two Tailed Test, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A and B ) Four days after being transfected with either pcDNA4/SPINK1 (SPINK1) or its EV, DU145 cells were subjected to qPCR. ( C – I ) HeLa/scramble cells and HeLa/shSPINK1-1, HeLa/shSPINK1-2, and HeLa/shSPINK1-3 cells were cultured under severe hypoxic conditions (O 2 < 0.1%) for 48 hours, irradiated with 0 ( C , D , and I ) or 4 ( E – I ) Gy of γ-rays and subjected to qPCR ( C – H ) or the DCFDA cellular ROS assay ( I ). Cells were irradiated in the presence or absence of the EGFR-I III ( G and H ). ( J ) DU145 cells were irradiated with γ-rays in the presence or absence of 100 ng/mL rSPINK1 in combination with DMSO or 2 μM ML385 and subjected to the colorimetric cell viability assay. Data are represented as mean ± SD ( n = 3 in A – J ). Two-tailed Student’s t test ( A , B , and J ). One-way ANOVA with Dunnett’s test ( C – I ). * P < 0.05, ** P < 0.01, *** P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; DCFDA, dichlorodihydrofluorescein diacetate; EGFR-I III, EGFR Inhibitor III.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Transfection, Cell Culture, Irradiation, ROS Assay, Viability Assay, Two Tailed Test, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A and B ) Two pairs of serial sections of clinical human ccRCC tissues from 2 independent patients were stained with the indicated antibodies in A and B , respectively. Corresponding serial sections were stained with H&E. Scale bar: 50 μm. N, necrosis. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; ccRCC, clear cell renal cell carcinoma.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Staining, Plasmid Preparation

Journal: JCI Insight

Article Title: SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization

doi: 10.1172/jci.insight.148135

Figure Lengend Snippet: ( A ) A scatter plot for correlation analysis between mRNA levels of SPINK1 and CA9 in 36 HeLa tumor xenografts showed a good coefficient of determination, R 2 = 0.9458. ( B and C ) After blood flow to the HeLa tumor xenografts was decreased by ligaturing the leg for the indicated times, levels of SPINK1 mRNA ( B ) and SPINK1 protein ( C ) in the tumor tissues were quantified by qPCR and the ELISA assay, respectively. ( D – I ) After anemia treatment by phenylhydrazine administration, mRNA levels of EPO in the kidneys ( D and E ) and those of CA9 ( E – H ) and SPINK1 ( F ) in tumor tissues were quantified by qPCR. SPINK1 protein levels in tumors ( G ) and plasma ( H and I ) and the tumor volume ( I ) were measured by the ELISA assay and digital calipers, respectively. Scatter plots for correlation analysis between the 2 indicated factors ( E – I ). Data are represented as mean ± SD ( B – D ; n = 36 in A , n = 9–10 in B and C , n = 5 in D – I ). Two-tailed Student’s t test ( D ). One-way ANOVA with Dunnett’s test ( B and C ). * P < 0.05, *** P < 0.001. SPINK1, serine peptidase inhibitor Kazal type 1; EV, empty vector; CA9, carbonic anhydrase 9; EPO, erythropoietin.

Article Snippet: Peripheral blood samples were centrifuged in EDTA tubes at 1200 g for 10 minutes at 4°C to quantify SPINK1 protein levels in plasma by the ELISA assay using Human SPINK1 DuoSet ELISA (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test, Plasmid Preparation