Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 expression is negatively correlated with TNBC prognosis. A) The UMAP plot in GSE176078 and t‐SNE plot in GSE75688 were performed to visualize cell types and SAT1 expression of each cell from breast cancer patients. B) The representative images of SAT1 expressions in different subtypes (n = 5) of breast cancer from SYSUCC using IHC. C) The bar plot of IHC scores in different subtypes of breast cancer from SYSUCC. D) Comparison of the SAT1 expression across normal tissues and TNBC tissues in GSE38959, GSE45827 and GSE65194. E–G) The SAT1 expressions were detected in paired tumor‐adjacent normal tissues and TNBC tissues (n = 4) through RT‐qPCR (E), western blots (F) and IHC (G). H,I) The representative IHC images (H) of various SAT1 expressions in TNBC and the Kaplan–Meier survival analysis (I) for OS performed in 100 TNBC patients based on different SAT1 levels.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Expressing, Comparison, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 knockdown inhibits TNBC progression in vivo. A) The in vivo bioluminescence imaging and tumor images of xenograft models generated by SAT1 WT and SAT1 KD cells (n = 5). B) The tumor growth curves of mice xenograft models generated by SAT1 WT and SAT1 KD cells. C) IHC staining of EMT‐related markers in tumor tissues derived from mice xenograft models in both SAT1 WT and SAT1 KD groups. D) The in vivo bioluminescence imaging (left) and luciferase activity analysis (right) of mice liver metastases models in both SAT1 WT and SAT1 KD groups (n = 3). E,F) The bioluminescence images and HE staining of mice liver metastases (E), and statistics on the number of liver metastatic nodules (F) in both SAT1 WT and SAT1 KD groups.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Knockdown, In Vivo, Imaging, Generated, Immunohistochemistry, Derivative Assay, Luciferase, Activity Assay, Staining

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: JUN boosts SAT1 transcription by binding to its promoter sequence. A) The top 10 transcription factors (TFs) binding to SAT1 promoter were obtained from the JASPAR database. B) The relationship between SAT1 expression and the top three transcription factors was determined with Spearman's correlation analyses in TNBC samples from TCGA‐BRCA cohort. C) The binding motif of JUN on SAT1 promoter. D) Four potential sites where JUN binds to SAT1 promoter were obtained from the JASPAR database. E,F) The alterations of SAT1 in response to JUN inhibition were detected by RT‐qPCR (E) and western blots (F), respectively. G) The dual‐luciferase reporter assay was used to confirm the binding of JUN to SAT1 promoter. H) The ChIP assay with JUN antibody was employed to verify the direct binding of JUN to SAT1 promoter. I) The dual‐luciferase reporter assay reaffirmed the key binding site of JUN to SAT1 promoter sequence.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Binding Assay, Sequencing, Expressing, Inhibition, Quantitative RT-PCR, Western Blot, Luciferase, Reporter Assay

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 stabilizes YBX1 protein through deubiquitylation. A) Identification of 79 interacted proteins of SAT1 through CoIP‐MS analyses in both BT549 and SUM159PT cells. B) The silver staining after immunoblotting of SAT1 in BT549 cells. C) The interaction between SAT1 and YBX1 was verified using CoIP followed by immunoblotting. D,E) Co‐localizations of SAT1 and YBX1 in both TNBC cells (D) and tissues (E) were visualized with immunofluorescence. F) The RT‐qPCR for YBX1 mRNA expression in SAT1 WT and SAT1 KD cells. G) Western blots for YBX1 protein expression in SAT1 WT and SAT1 KD cells. H) Immunofluorescence images for YBX1 protein in SAT1 WT and SAT1 KD cells. I) Western blots for SAT1 in SAT1 KD cells with or without YBX1 overexpression. J) Western blots for YBX1 at different times (left) and quantization of half‐life time (right) of YBX1 in SAT1 WT and SAT1 KD cells in response to CHX (50 µ m ) treatment. K) Western blots for YBX1 in SAT1 WT and SAT1 KD cells after MG132 (50 µ m ) treatment for 12 h. L) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells transfected with the indicated plasmids to detect the alteration of HA‐YBX1 ubiquitylation level.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Silver Staining, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing, Over Expression, Transfection

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: HERC5 mediates YBX1 ubiquitination induced by SAT1 knockdown. A) The protein mass spectrometry data of YBX1 immunoprecipitants from BT549 cells. B) Co‐immunoprecipitation for indicated protein followed by immunoblots for HERC5 in BT549 cells. C) Co‐localization of HERC5 and YBX1 in BT549 cells was visualized with immunofluorescence. D) Western blots for YBX1 in BT549 cells with HERC5 inhibition or overexpression. E) Western blots for YBX1 in TNBC cells with overexpressed HERC5 in response to CHX (50 µM) treatment for different periods. F,G) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells with HERC5 inhibition (F) or overexpression (G). H) Immunoblots of cell lysate and HA‐tagged immunoprecipitants from HEK293T cells with inhibition of both SAT1 and HERC5.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Ubiquitin Proteomics, Knockdown, Mass Spectrometry, Immunoprecipitation, Western Blot, Immunofluorescence, Inhibition, Over Expression

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 deficiency activates autophagy in TNBC. A) The Venn diagram showed pathway enrichment analyses of SAT1 in three TNBC cohorts (GSE38959, GSE45827 and GSE65194). B) The GSEA results of autophagy in the above three datasets. C,D) Western blots for LC3 and p62 in SAT1 WT or SAT1 KD cells after EBSS treatment for indicated hours (C); The fold change of LC3 II was calculated by bar plot (D). E) Western blots for LC3 and p62 in SAT1 WT or SAT1 KD cells with or without BafA1 (100 n m ) treatment for 12 h. F) Western blots for LC3 and p62 in indicated cells with or without chloroquine treatment for 12 h. G,H) SAT1 WT and SAT1 KD BT549 cells transfected with an mRFP‐GFP‐LC3 reporter were treated with or without BafA1 (100 n m ) for 12 h. Representative confocal images were presented G), and the number of autophagosomes (yellow puncta) and autolysosomes (red puncta) per cell was quantified H). I) Western blots for LC3 and p62 in SAT1 KD cells with or without Baf‐A1 (100 nM) treatment in response to YBX1 overexpression. J) The representative confocal images of mRFP‐GFP‐LC3 assay in SAT1 WT and SAT1 KD BT549 cells which were transfected with or without YBX1 overexpression and treated with or without Baf‐A1 (100 nM) for 12 h.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Western Blot, Transfection, Over Expression

Journal: Advanced Science

Article Title: Autophagy Deficiency Induced by SAT1 Potentiates Tumor Progression in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202309903

Figure Lengend Snippet: SAT1 suppresses autophagy through YBX1‐mediated m5C modification of mTOR mRNA. A) The GSEA results of mTOR signaling pathway in a TNBC cohort‐GSE38959. B) Western blots for mTOR and its downstream effectors in SAT1 WT and SAT1 KD cells. C) Western blots for mTOR and its downstream effectors in SAT1 KD cells with or without overexpressed YBX1. D) qPCR for the mTOR mRNA expression in indicated cells with knockdown of SAT1 or YBX1. E) mRNA quantification of mTOR in BT549 cells with YBX1 knockdown or overexpression in response to actinomycin D treatment for indicated hours. F) mRNA quantification of mTOR in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression in response to actinomycin D treatment. G) The RIP‐qPCR assay to detect the binding between YBX1 and mTOR mRNA in BT549 cells. H) Lysates of BT549 cells were pulled down with biotinylated probe recognizing mTOR mRNA, and then YBX1 in the precipitates was detected by western blots. I) Dot blot assay for m5C levels in BT549 cells with YBX1 knockdown or overexpression. The intensity of dot immunoblotting (above) represented the m5C levels while methylene blue staining (below) indicated the amount of loaded RNA. J) Dot blot assay for m5C levels in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression. K) The RIP‐qPCR assay to verify the binding between m5C and mTOR mRNA in BT549 cells. L) The RIP‐qPCR assay to qualify m5C enrichment levels of mTOR mRNA in SAT1 WT and SAT1 KD BT549 cells with or without YBX1 overexpression. (M) A schematic of the conclusion in this current study.

Article Snippet: Notably, plasmids of human SAT1 promoter wildtype and mutants were all generated with a pEZX‐FR01 vector (GeneCopeia, USA).

Techniques: Modification, Western Blot, Expressing, Knockdown, Over Expression, Binding Assay, Dot Blot, Staining

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 1. Arginine is elevated in liver tumors and promotes tumor formation (A) Hierarchical clustering of significantly altered metabolites from control (Ctrl) liver and tumor tissues (T) from liver-specific Tsc1 and Pten double-knockout (hereafter, L-dKO) mice. n = 5 (Ctrl), n = 6 (L-dKO). (B) Up- and downregulated metabolic pathways in L-dKO tumors compared to Ctrl liver tissues, summarized from MPWEA (see Table S1). (C) Amino acid profile of L-dKO tumor relative to Ctrl liver tissues (log2 ratio). n = 5. (D) Schematic representation of arginine and polyamine metabolism. Boxes below enzymes indicate changes in mRNA (left box) and protein (right box) levels in L-dKO tumors compared to Ctrl livers, respectively. Color coding according to level of log2-fold change as indicated. SMOX, spermine oxidase; SAT1, sper- midine/spermine N-acetyltransferase 1; PAOX, polyamine oxidase; ‘‘?’’ indicates unknown identity. n = 6 (Ctrl), n = 12 (L-dKO). (E) Immunoblots of arginine-synthesizing enzymes (CPS1, OTC, ASS1, and ASL) and arginine transporters (SLC7A1, SLC7A6, and SLC7A7) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control. n = 4 (Ctrl), n = 8 (L-dKO). (F) Relative 3H-arginine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (G) Number of macroscopic tumors per liver of L-dKO mice fed diets containing standard content (100%), 10%, or 1% of arginine for 8–20 weeks of age. n = 6–9. (H) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice fed with arginine-modified diets. n = 3–9. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired t test (C and F) and one-way ANOVA (G and H).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Control, Double Knockout, Western Blot

Journal: Cell

Article Title: Arginine reprograms metabolism in liver cancer via RBM39.

doi: 10.1016/j.cell.2023.09.011

Figure Lengend Snippet: Figure 2. Loss of ARG1 and AGMAT enhances liver tumor formation (A) Immunoblots of arginine-to-polyamine-converting enzymes (ARG1 and AGMAT) and polyamine metabolism enzymes (ODC, SRM, SMS, SAT1, PAOX, and SMOX) in Ctrl liver and L-dKO tumor tissues. Calnexin serves as loading control (same samples were used as in Figure 1E). n = 4 (Ctrl), n = 8 (L-dKO). (B) Total polyamine content in Ctrl liver and L-dKO tumor tissues. n = 6. (C) Relative 3H-putrescine uptake into Ctrl liver and L-dKO tumor tissues. n = 8. (D) Immunohistochemistry of Ctrl and L-dKO liver tissues stained for ARG1 or AGMAT. NT, adjacent non-tumor tissue; T, tumor. (E) Representative images of livers from L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. (F) Number of macroscopic tumors per liver of L-dKO mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 9–10. (G) Arginine content in Ctrl liver and L-dKO non-tumor (NT) and tumor (T) tissues of mice injected with AAV-Ctrl, AAV-ARG1, or AAV-AGMAT. n = 4–10. *p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001 by unpaired t test (B and C) and one-way ANOVA (F and G).

Article Snippet: Antibodies used in this study were as follows: ARG1 (GeneTex, Cat# 109242), AGMAT (Novus Biological, Cat# 1–82080), CPS1 (abcam, Cat# 129076), OTC (SantaCruz Biotech, Cat# 515791), ASS1 (SantaCruz Biotech, Cat# 365475), ASL (SantaCruz Biotech, Cat# 166787), SLC7A1 (abcam, Cat# 37588), SLC7A6 (MyBiosource, Cat# 7103267), SLC7A7 (Epigentek, Cat# A68118-020), ODC (GeneTex, Cat# 54600), SRM (ThermoFisher Scientific, Cat# PA5-31341), SMS (SantaCruz Biotech, Cat# 376294), SAT1 (Novus Biological, Cat# 110–41622), PAOX (SantaCruz Biotech, Cat# 166185), SMOX (abcam, Cat# 213631), AKT (Cell Signaling, Cat# 4685), AKT-pS473 (Cell Signaling, Cat# 9217), Calnexin (Enzo Life Sciences, Cat# ADI-SPA-860-F), Actin (Millipore, Cat# MAB1501), ASNS (GeneTex, Cat# 30068), PSAT1 (GeneTex, Cat# 633629), PSPH (GeneTex, Cat# 33442), NNMT (abcam, Cat# 119758), S6-pS240,244 (Cell Signaling, Cat# 5364), S6 (Cell Signaling, Cat# 2217), RBM39 (Sigma, Cat# HPA001591), RBM39 (Bethyl Laboratories, Cat# A300-291A), FLAG M2 (Sigma, Cat# F1804), HA (Cell Signaling, Cat# 2367), Strep (Invitrogen, Cat# MA5-37747), eIF2a (Cell Signaling, Cat# 2103), eIF2a-pS51 (Cell Signaling, Cat# 3957), SESN2 (abcam, Cat# ab178518), CASTOR1 (SantaCruz Biotech, Cat# 377114), H3 (Cell Signaling, Cat# 14269), GAPDH (SantaCruz Biotech, Cat# 365062).

Techniques: Western Blot, Control, Immunohistochemistry, Staining, Injection

Journal: Communications Biology

Article Title: Nasal symbiont Staphylococcus epidermidis restricts influenza A virus replication via the creation of a polyamine-deficient cellular environment

doi: 10.1038/s42003-024-06706-4

Figure Lengend Snippet: A Schematic mechanism of the depletion of polyamines by DENSpm, B SAT1 protein expression, C Alterations of polyamine metabolism-related metabolites, D , E viral PA mRNA expression by treating DENSpm or synthetic polyamines, and F viral NP protein expression. DENSpm-N1 N11-diethylnorspermine, SAT1 spermidine/spermine N1, acetyltransferase, PA polymerase, NA neuraminidase.

Article Snippet: Human ODC1 antibody (molecular weight 61 kDa, cat# MAB2695-SP, primary antibody 1:500), anti-β-actin antibody (molecular weight 43 kDa, primary antibody 1:500), and human SAT1 antibody (molecular weight 20 kDa, cat #AF-1786) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Nature communications

Article Title: Promotion of homology-directed DNA repair by polyamines.

doi: 10.1038/s41467-018-08011-1

Figure Lengend Snippet: Fig. 2 Polyamines affect DSB repair by homologous recombination. a Schematic of the DR-GFP reporter assay to assess HR activity. b, c U2OS cells were treated with 500 μM DFMO for the indicated times. b Levels of three polyamines were quantified and normalized to that in untreated cells. Note that, in agreement with previous studies7,14, treatment with DFMO significantly attenuated levels of putrescine and spermidine but had a lesser effect on that of spermine. c Determination of HR efficiency using the DR-GFP reporter assay. GFP+ cells were quantified by flow cytometry 48 h after I-SceI transfection. Relative HR frequency was normalized to the percentage of untreated cells. Expression levels of I-SceI and tubulin were examined by immunoblotting. d, e U2OS cells were transfected without (Ctrl) or with scrambled siRNA (SCR), ODC siRNA, or expression vector of SAT1 for 24 h. The cells were harvested for analyses 48 h after I-SceI transfection. d The level of individual polyamine was quantified and normalized to that in untransfected cells. e HR efficiency was determined using the DR-GFP reporter assay. The percentage of GFP+ cells was normalized to untransfected cells. Expression levels of ODC and tubulin were examined by immunoblotting. f, g The effects of DFMO (500 µM) treatment and ODC overexpression on HR efficiency were examined in U2OS cells. Untreated cells without any treatment or transfection were included as the control (Ctrl). f The relative level of individual polyamine was determined 2 days after overexpression of ODC. g HR efficiency was quantified by flow cytometry and normalized to control cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant (P > 0.05). Data are the mean ± s.e.m. from three independent experiments (n = 3). Statistics was performed by one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file

Article Snippet: The human SAT1 (RefSeq NM_002970.1) expression plasmid was purchased from OriGene.

Techniques: Homologous Recombination, Reporter Assay, Activity Assay, Cytometry, Transfection, Expressing, Western Blot, Plasmid Preparation, Over Expression, Control

Journal: Cancers

Article Title: Disulfiram/Copper Induces Antitumor Activity against Both Nasopharyngeal Cancer Cells and Cancer-Associated Fibroblasts through ROS/MAPK and Ferroptosis Pathways

doi: 10.3390/cancers12010138

Figure Lengend Snippet: DSF/Cu inhibits nasopharyngeal carcinoma cells via the ferroptosis pathway. ( A ) Western blot analysis for the expression of p53-related proteins in 5-8F treated with DSF/Cu (1 μM/1 μM); the p53 inhibitor Pifithrin-α (20 μM) was pretreated for 12 h. Data are shown as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3. ( B , C ) RT-qPCR analysis of Ptgs2 and SAT1 mRNA levels in 5-8F. Data are shown as means ± SD. * p < 0.05, *** p < 0.001 vs. control group, n = 3. ( D ) The expression of SAT1 was detected by Western blotting in 5-8F, after cultured with DSF/Cu (1 μM/1 μM). Data are shown as means ± SD. *** p < 0.001, n = 3. ( E ) RT-qPCR analysis of ALOX15 mRNA levels in 5-8F. Data are shown as means ± SD. ** p < 0.01, *** p < 0.001 vs. control group, n = 3. ( F ) Lipid ROS production in 5-8F treated with DSF/Cu (1 μM/1 μM) for 6 h was assessed by flow cytometry using C11-BODIPY. NAC was pretreated for 12 h. Data are shown as means ± SD. *** p < 0.001, n = 3. ( G ) The percentage of viable 5-8F cells after being treated with DSF/Cu (1 μM/1 μM) for 24 h. Inhibitors were pretreated for 12 h. DMSO solvent was used as a control. Data are shown as means ± SD. *** p < 0.001 vs. DSF/Cu group (the second group), n = 3.

Article Snippet: Total cell proteins were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies directed against human PARP, Caspase3, Cleaved-Caspase3, p-JNK, p38, p-p38 and BAX (1:1000, Cell Signaling Technologies, CST, Boston, MA, USA), JNK (1:500, Wanleibio, Shenyang, China), p53 and p21 (1:1000, proteintech, Chicago, IL, USA), ALDH1A1 and ALDH2 (1:100, Boster, Wuhan, China), SAT1 (1:500, Elabscience, Wuhan, China), α-SMA (1:100, Boster, Wuhan, China), and FAP-α (1:400, R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Cell Culture, Flow Cytometry

Journal: Nutrition & Metabolism

Article Title: Untargeted metabolomic analysis of ischemic injury in human umbilical vein endothelial cells reveals the involvement of arginine metabolism

doi: 10.1186/s12986-023-00737-0

Figure Lengend Snippet: Arginine metabolism pathway-related proteins changed significantly in HUVECs after different OGD treatment periods (0, 3, 6 and 9 h). A – D Intracellular mRNA levels of ASS1, ARG2, ODC1 and SAT1 were measured by RT‒PCR, n = 3/group. E Western blotting results of four metabolism-related proteins (ASS1, ARG2, ODC1 and SAT1), n = 3/group. F Quantitative analysis results of the four proteins. One-way ANOVA with multiple comparisons was utilized to determine the statistical significance as follows: * p value < 0.05, ** p value < 0.01, and *** p value < 0.001

Article Snippet: The following antibodies were used for western blotting: rabbit monoclonal anti-cleaved caspase 3 (Asp175) antibody (#9664S, Cell Signaling Technology, 1:1000); mouse monoclonal anti-β-actin antibody (#3700S, Cell Signaling Technology, 1:1000); rabbit monoclonal anti-GAPDH antibody (#5174S, Cell Signaling Technology, 1:1000); rabbit monoclonal anti-ASS1 (#70720S, Cell Signaling Technology, 1:1000); rabbit monoclonal anti-SAT1 (#61586S, Cell Signaling Technology, 1:1000); rabbit monoclonal anti-ODC1 antibody (ab270268, Abcam, 1:1000); rabbit polyclonal anti-ARG2 antibody (ab264066, Abcam, 1:1000); goat anti-rabbit IgG antibody (ab6721, Abcam, 1:5000); and goat anti-mouse IgG antibody (ab6789, Abcam, 1:5000).

Techniques: Western Blot