Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, Histogram of relative abundance of 3160 proteins detected (out of 5399 total, see ) by LC–MS/MS in 8.7 × 10 4 purified human ²-cells from n = 3 donors. Green lines indicate gene products related to the urea cycle, argininosuccinate and aspartate metabolism detected at the protein level. b, Expression levels of genes related to the urea cycle, pyruvate metabolism and arigninosuccinate shunt based on RNAseq analysis of FACS-sorted human β-cells from n=3 human donors, data are in Log2CPM. c, Co-immunostaining of insulin and individual metabolic enzymes related to the urea cycle in dispersed human islet cells from one donor representing similar results obtained from two additional donors. Representative images are shown (left), scale bar is 10 microns. Mean fluorescence intensity (FI) within the insulin positive region of interest (ROI) was calculated from 5 images per antibody (right). Neg denotes negative control for background Alexa Fluor 488 signal with insulin co-stain. Statistical analyses are student’s t-tests of each enzyme compared to Neg. Enzyme abbreviations in a–c are ARG2, arginase 2; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthase 1; CPS1, carbamoyl-phosphate synthase 1; DDAH 1, dimethylarginine dimethylaminohydrolase 1; DDAH 2, dimethylarginine dimethylaminohydrolase 2; GOT1, aspartate aminotransferase 1; GOT2, aspartate aminotransferase 2; MDH1, malate dehydrogenase 1; MDH2, malate dehydrogenase 2; NAGS, N-acetyl-glutamate synthase; OAT, ornithine aminotransferase; PC, pyruvate carboxylase; SLC25A12, solute carrier family 25 member 12/calcium-binding mitochondrial carrier protein Aralar 1; SLC25A13, solute carrier family 25 member 13/calcium-binding mitochondrial carrier protein Aralar 2; SLC25A15, solute carrier family 25 member 15/mitochondrial ornithine transporter 1. d–e, Cytokine-induced changes in the partitioning of 15 N 2 -L-arginine to urea ( d ) and citrulline/NO ( e ) synthesis in human islets comparing protective vs non-protective glucose metabolism as modeled by BAD SAHB A SD vs RO0281675 treatment, respectively, n=4 donors. f, Chemical inhibition of arginase via ABH interferes with the protective effect of BAD SAHB A SD in human islets undergoing inflammation, n=5 donors. Data are means ± s.e.m. with one-way (d,e) and two-way (f) ANOVA statistical tests with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Purification, Expressing, Immunostaining, Fluorescence, Negative Control, Staining, Binding Assay, Inhibition

Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, Schematic of the TCA and urea cycles and their connection via the aspartate-argininosuccinate shunt. Enzymes of interest are marked in red and their corresponding inhibitors in blue. b, Total aspartate levels in human islets treated with the indicated compounds and cultured in the absence or presence of inflammatory cytokines. Data are from the untargeted metabolomics analysis in , n=5 human donors pooled and split into 8 replicates. c, Contribution of glucose to total aspartate pools in mouse islets labeled with 13 C 6 glucose and treated with inflammatory cytokines in the context of protective vs non-protective glucose metabolism. Data are from n=5 (Veh, RO0281675), n=6 (BAD SAHB A SD) and n=4 (BAD SAHB A AAA) independent mouse islet isolations and experiments. See for isotopologue distribution of aspartate in an analogous labelling experiment. d–e, Quantification of urea and NO , and viability ( e ) in human islets subjected to shRNA-mediated GOT1 ( G1 ) or GOT2 ( G2 ) depletion and treated with cytokines in the context of protective vs non-protective glucose metabolism, n=4 donors for urea, n=3 donors for NO, and n=5 donors for viability measurements. Data for one hairpin per gene are displayed (sh#1 for GOT1 and sh#2 for GOT2 ) from the full data set of multiple hairpins, see – . f–g, Quantification of urea levels ( f ) and viability ( g ) in human islets supplemented with argininosuccinate (AS) in the presence of inflammatory cytokines, H 2 O serves as vehicle control for AS. Data are from 6 independent experiments from n=3 donors. Data are means ± s.e.m. with statistical analyses on means from independent experiments using one-way ANOVA with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Cell Culture, Labeling, shRNA

Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, 13 C fractional labelling of aspartate from 13 C 6 glucose. Data are shown as non-normalized to vehicle PBS and display the fraction of each M+ n mass isotopomer out of the total pool of aspartate for each condition. For clarity, statistical comparisons are only shown for each M+ n of a given condition (RO0281675, BAD SAHB A SD and BAD SAHB A AAA) compared to the corresponding M+ n of vehicle control. Data are pooled means from n=6 (Veh), n=5 (RO0281675), and n=6 (BAD SAHB A SD, BAD SAHB A AAA) independent mouse islet isolations and experiments. b, Western blot analysis of GOT1/2 knockdown efficiency using multiple independent hairpins for data shown in – and – . Blots are representative of n=2 independent experiments with similar results. c–d, Aspartate ( c ), urea and NO ( d ) levels in human islets from the same experiments shown in – , displaying the complete set of data on all hairpins tested. Aspartate data are from n = 4 human donors for shCtrl samples and n = 3 donors for knockdown samples. Urea and NO data are from n = 4 and n = 3 donors, respectively. Statistical analyses in (a) are two-way ANOVA showing p-value comparisons for each condition to Veh, and one-way ANOVA in (c–d), both with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Western Blot

Journal: Cancer research

Article Title: Nitrogen trapping as a therapeutic strategy in tumors with mitochondrial dysfunction

doi: 10.1158/0008-5472.CAN-20-0246

Figure Lengend Snippet: (Left panel) Under OXPHOS-competent conditions, aspartate is normally produced by GOT2 in the mitochondria. dmaKG can increase intracellular aKG levels and promote mitochondrial metabolism to maintain aspartate levels. (Right panel) Under OXPHOS-incompetent conditions, cells rely on GOT1 in the cytosol for aspartate synthesis. Aberrant elevation in aKG levels can result in nitrogen trapping (converting aKG to Glu) via GOT1, leading to aspartate exhaustion. Blue arrows depict the mode of action of dmaKG. Glu, glutamate; OAA, oxaloacetate.

Article Snippet: Chandel (Northwestern University), and they were cultured in the standard medium supplemented with 0.1 mg/mL uridine ( 17 ). pMXs-GOT1 and pMXs-IRES-blasticidin empty vector were introduced into UOK262, 143B-wt and 786-O by viral transduction to generate stable lines. pMXs-SLC1A3 (Addgene, #72873) and pMXs-GOT1 (Addgene, #72872) were gifts from David Sabatini ( 15 ).

Techniques: Produced

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p, sponged by NEAT1, overexpressed in vitro and in vivo and might promote cell apoptosis in sepsis-induced ferroptosis by targeting TFRC and GOT1 A The binding regions between miR-9-5p with sequences from NEAT1, TFRC , and GOT1 . B , C The expression level of miR-9-5p, TFRC , and GOT1 in model and control was tested by qRT-PCR in vivo (normalized to U6 or GAPDH ). *** P < 0.001 vs control. D The protein levels of TFRC and GOT1 in cerebral cortex were assessed using Western blotting. E The protein level of TFRC in cerebral cortex was showed by immunohistochemistry. F , G The dual-luciferase reporter gene assay analyzes the binding relationship between miR-9-5p with sequences from NEAT1, TFRC , and GOT1 . *** P < 0.001 vs NC. H , I The expression level of miR-9-5p, TFRC , and GOT1 in iron-rich (100 μM FeCl 3 ), control (100 μM FeCl 3 + control serum), and model (100 μM FeCl 3 + sepsis serum) group was tested by qRT-PCR (normalized to U6 or GAPDH ). * P < 0.05, ** P < 0.01, *** P < 0.001 vs iron-rich and control. J The protein levels of TFRC and GOT1 assessed using western blotting in vitro. K , L The immunofluorescence for detecting TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1).

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: In Vitro, In Vivo, Binding Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Luciferase, Reporter Gene Assay, Immunofluorescence

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Increased miR-9-5p regulates the expression of TFRC and GOT1 and relieves ferroptosis in vitro The bEnd.3 cells were transfected with miR-9-5p angomir, followed by serum stimulation. A The expression levels of NEAT1, miR-9-5p, TFRC , and GOT1 in control, model, and model + miR-9-5p angomir were tested by qRT-PCR (normalized to U6 or GAPDH ). B The sequence for NEAT1 mutant. C The protein levels of TFRC and GOT1. D , E The immunofluorescence for TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1). F The cell viability of bEnd.3 cells were evaluated by CCK-8 assay. G – K The levels of ROS, Fe ion (Fe 2+ and Fe 3+ ), GSH, GPX4, and MDA were assessed. * P < 0.05, ** P < 0.01, *** P < 0.001 vs control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs model.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vitro, Transfection, Control, Quantitative RT-PCR, Sequencing, Mutagenesis, Immunofluorescence, CCK-8 Assay

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: Enhanced NEAT1 promoted ferroptosis and increased the expression of TFRC and GOT1 in vitro NEAT1 was overexpressed in bEnd.3 cells. A The expression levels of NEAT1, miR-9-5p, TFRC , and GOT1 in Vector, WT NEAT1 (wild NEAT1 sequence), and MUT NEAT1 (site-directed mutant NEAT1) by qRT-PCR (normalized to U6 or GAPDH ). B The protein levels of TFRC and GOT1 showed by western blotting. C , D The immunofluorescence for TFRC and GOT1 (blue, DAPI; green, TFRC; red, GOT1). E The cell viability of cells detected by CCK-8 assay. F – J The levels of ROS, Fe ion (Fe 2+ and Fe 3+ ), GSH, GPX4, and MDA. ** P < 0.01, ** P < 0.01, *** P < 0.001 vs Vector; ## P < 0.01, ### P < 0.001 vs WT NEAT1.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vitro, Plasmid Preparation, Sequencing, Mutagenesis, Quantitative RT-PCR, Western Blot, Immunofluorescence, CCK-8 Assay

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p suppressed the expression of TFRC and GOT1 in vivo A – D The qRT-PCR analysis on the expression of NEAT1, miR-9-5p, TFRC , and GOT1 in the cerebral cortex of control, model, and model + miR-9-5p angomir rats, respectively (normalized to U6 or GAPDH ). E The western blotting analysis on TFRC and GOT1. F The protein level of TFRC in four brain regions were assessed by Immunohistochemistry. *** P < 0.001 vs control; ### P < 0.001 vs model.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vivo, Quantitative RT-PCR, Control, Western Blot, Immunohistochemistry

Journal: Molecular Neurobiology

Article Title: Exosome-Derived lncRNA NEAT1 Exacerbates Sepsis-Associated Encephalopathy by Promoting Ferroptosis Through Regulating miR-9-5p/ TFRC and GOT1 Axis

doi: 10.1007/s12035-022-02738-1

Figure Lengend Snippet: miR-9-5p suppressed the expression of and GOT1 in vivo The protein level of GOT1 in four brain regions was assessed by Immunohistochemistry.

Article Snippet: Subsequently, the samples was incubated with primary antibodies, including rabbit anti-mouse polyclonal antibodies to TSG01 (1:2000; ab125011), CD9 (1:2000; ab92726), CD63 (1:2000; ab193349), TFRC (1:2000; ab214039), GOT1 (1:1000; ab221939) and GAPDH (1:5,000; ab8245) for 1 h. The samples were then incubated in the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:20,000, BA1054, BOSTER Inc.) with oscillation at room temperature for 40 min.

Techniques: Expressing, In Vivo, Immunohistochemistry

Journal: Archives of Toxicology

Article Title: PET microplastics alter the transcriptome profile and oxidative stress markers in the liver of immature piglets: an in vivo study

doi: 10.1007/s00204-025-04151-8

Figure Lengend Snippet: The effect of low (LD) and high (HD) doses of microplastics on liver function and fibrosis-related parameters in tissue homogenates, measured using ELISA. The concentrations of bilirubin, collagen type IV, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) are presented as mean ± SEM. The asterisks indicate statistically significant differences ( p ≤ 0.05) compared to the control group (C)

Article Snippet: The protein concentrations of total bilirubin (Porcine Total bilirubin ELISA Kit, Cat. No EA0061Po, BT LAB, China), collagen type IV (Porcine Collagen Type 4 ELISA Kit, Cat. No E0020Po, BT LAB, China), alanine aminotransferase (ALT, Porcine Alanine Aminotransferase ELISA Kit, Cat. No E0150Po, BT LAB, China), and aspartate aminotransferase (AST, Porcine Aspartate Aminotransferase, cytoplasmic ELISA Kit, Cat. No E0529Po, BT LAB, China) in tissue homogenates were determined using a commercial ELISA kit specific for pigs, following the manufacturer’s protocol as previously described.

Techniques: Enzyme-linked Immunosorbent Assay, Control