Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1

doi: 10.1007/s00018-023-05043-9

Figure Lengend Snippet: SIRT1 inhibited GOT1 enzyme activity by catalyzing its lysine deacetylation. A The concentration of Asp in the femurs of the control and cKO mice aged 8 weeks measured by an ELISA Kit (n = 8/group). B The concentration of malate in the femurs of the control and cKO mice aged 8 weeks measured by an ELISA Kit (n = 8/group). C The activity of GOT1 in the femurs of the control and cKO mice aged 8 weeks measured by an ELISA Kit (n = 8/group). D The subcellular localization of SIRT1 and GOT1 in primary osteoblasts. E The protein level and Kac level of GOT1 upon Sirt1 deletion or overexpression in 3T3E1 cells. F The Kac level of GOT1 in Sirt1 knockout 3T3E1 cells upon Sirt1 overexpression. G The Kac level of GOT1 in 3T3E1 cells treated with SRT2104 or EX-527. H The Kac level of GOT1 protein immunoprecipitated from 3T3E1 cells transfected with SIRT1 plasmids or mutant SIRT1-H355A plasmids. I Control and Sirt1 knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The Kac level of GOT1 protein immunoprecipitated from the above cells. J Sirt1-knockout/Got1- knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The GOT1 activity was measured in the above cells. K Sirt1-knockout/Got1- knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The levels of F6P in glycolysis were measured in the above cells. L Sirt1-knockout/Got1- knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The levels of PEP in glycolysis were measured in the above cells. M Sirt1-knockout/Got1- knockout 3T3E1 cells were infected with Wild-type GOT1 or GOT1-K318R mutant virus. The ALP staining of osteoblast mineralization was conducted in the above cells. Data are presented as the mean ± SD, Student’s t test (a, b, c); one-way ANOVA with Tukey’s test (j, k, l), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: GOT1 activity inhibition To inhibit the activity of GOT1, mice aged 8 weeks were treated with vehicle (saline) and/or 10 mg/kg aminooxyacetic acid (AOA) (MedChemExpress, HY-107994) every other day until dissection at 16 weeks, and osteoblasts were treated with vehicle or 100 μM AOA for 48 h. Statistics Statistical analyses were performed using GraphPad Prism 9.0.

Techniques: Activity Assay, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Knock-Out, Immunoprecipitation, Transfection, Mutagenesis, Infection, Virus, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1

doi: 10.1007/s00018-023-05043-9

Figure Lengend Snippet: SIRT1 affected the acetylation level of GOT1 and glycolysis to regulate bone mass. A Schematic of experimental design. The 8-week cKO mice treated with vehicle or AOA until dissection at 16 weeks. B GOT1 activity of 16-week cKO mice treated with vehicle or AOA (n = 8/group). C The concentration of Asp in 16-week cKO mice treated with vehicle or AOA (n = 8/group). D The concentration of malate in 16-week cKO mice treated with vehicle or AOA (n = 8/group). E Representative micro-CT images of the femurs in 16-week cKO mice treated with vehicle or AOA. F Quantification of the BV/TV, Tb.N, Tb.Th, and Tb.Sp from the distal femurs in 16-week cKO mice treated with vehicle or AOA (n = 7/group). G Quantification of the metabolites in glycolysis after AOA treatment in 16-week cKO mice (n = 8/group). H Relative mRNA expression of Runx2, Col1a1, Alpl, and Bglap in Sirt1KO osteoblasts treated with vehicle or AOA (n = 6/group). I Representative images of ALP staining of Sirt1KO osteoblasts treated with vehicle or AOA. J Representative images of Alizarin Red S staining of Sirt1KO osteoblasts treated with vehicle or AOA. K Quantification of caspase-3 activity in Sirt1KO osteoblasts treated with vehicle or AOA (n = 6/group). BV/TV: bone volume fraction; Tb.Th: trabecular thickness; Tb.N: trabecular number; Tb.Sp: trabecular separation; Cort.Th: cortical bone thickness; Tt.Ar: total cortical bone area. Data are presented as the mean ± SD, Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: GOT1 activity inhibition To inhibit the activity of GOT1, mice aged 8 weeks were treated with vehicle (saline) and/or 10 mg/kg aminooxyacetic acid (AOA) (MedChemExpress, HY-107994) every other day until dissection at 16 weeks, and osteoblasts were treated with vehicle or 100 μM AOA for 48 h. Statistics Statistical analyses were performed using GraphPad Prism 9.0.

Techniques: Dissection, Activity Assay, Concentration Assay, Micro-CT, Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1

doi: 10.1007/s00018-023-05043-9

Figure Lengend Snippet: Schematic diagram of SIRT1 deacetylating GOT1 to regulate glycolysis in osteoblasts. SIRT1 deletion induced acetylation of GOT1, thereby inhibited the process of glycolysis to decrease bone formation by osteoblasts. The decreased bone formation then inhibited bone resorption. Thus, bone mass decreased

Article Snippet: GOT1 activity inhibition To inhibit the activity of GOT1, mice aged 8 weeks were treated with vehicle (saline) and/or 10 mg/kg aminooxyacetic acid (AOA) (MedChemExpress, HY-107994) every other day until dissection at 16 weeks, and osteoblasts were treated with vehicle or 100 μM AOA for 48 h. Statistics Statistical analyses were performed using GraphPad Prism 9.0.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: GOT1 protein levels are regulated by the ubiquitin-proteasome pathway (A) A schematic of the malate-aspartate shuttle (MAS) in brown adipocytes. MAS consists of two mitochondrial transporters and two pairs of cytosolic (GOT1 and MDH1) and mitochondrial (GOT2 and MDH2) enzymes. Cold-activated MAS facilitates the transfer of reducing equivalents (especially, electrons from NADH) from the cytosol into the mitochondria. The illustration was created using BioRender ( https://www.biorender.com ). (B) qPCR analysis of Got1 gene expression in BAT during cold exposure. C57BL/6J female mice were housed at 23°C or exposed to 4°C for the times indicated. (C) GOT1 protein expression in cold-exposed BAT. (D) GOT enzymatic activity in BAT homogenates. (E) Degradation of GOT1 protein expressed in HEK293 cells upon treatment with 50 μg/mL of cycloheximide (CHX). A line graph shows the normalized GOT1 protein levels at the indicated time points from three independent experiments. (F) Co-treatment with MG132 attenuates proteasomal degradation of GOT1 in the presence of cycloheximide (CHX). (G) Ubiquitination of GOT1 protein immunoprecipitated from HEK293 cells treated with 10 µM of MG132 for 2 h. (H) Accumulation of GOT1 protein levels in brown adipocytes upon treatment with 10 µM of MG132 for 8 h. Brown adipocytes were stimulated with the β-adrenergic receptor agonist isoproterenol (ISO). All data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Ubiquitin Proteomics, Gene Expression, Expressing, Activity Assay, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: RNF34 is an E3 ubiquitin ligase for GOT1 (A) A schematic describing the ubiquitin-proteasome-dependent degradation of the protein. E2: E2 ubiquitin-conjugating enzyme, E3: E3 ubiquitin ligase. (B) RNF34-dependent ubiquitination and degradation of GOT1 in HEK293 cells. (C) Rnf34 gene expression in BAT from mice exposed to 4°C for the times indicated. (D) RNF34 protein levels in BAT from mice housed at 23°C or 4°C for 7 days. All data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Ubiquitin Proteomics, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: The βAR-SGK1 signaling pathway increases GOT1 protein stability in brown adipocytes (A) Effect of kinase inhibitors on GOT1 protein levels. Isoproterenol (ISO)-treated brown adipocytes were co-treated for 2 h with KU0063794, GSK690693, GSK650394, and Go 6,983 to inhibit mTORC2, Akt, SGK, and PKC, respectively. (B) No alteration of Got1 gene expression by the SGK inhibitor GSK650394 (25 µM). (C) Effect of cold stress on the expression of Sgk1 , Sgk2 , and Sgk3 isoforms in BAT. C57BL/6 male mice were housed at 23°C or exposed to 4°C. (D) SGK1 activation stabilizes GOT1. HEK293 cells co-expressing GOT1 and SGK1 S422D or SGK2 S356D were treated with 50 μg/mL of CHX. (E) Sgk1 expression in brown adipocytes treated with vehicle or 10 µM of isoproterenol (ISO) for 4 h. (F) SGK1 S422D -dependent phosphorylation of NDRG1 at Thr346 in HEK293 cells in the absence and presence of MG132. (G) Accumulation of SGK1 protein in brown adipocytes during treatment with 10 µM of MG132. (H) β-adrenergic stimulation of brown adipocytes with isoproterenol induces phosphorylation of NDRG1 at Thr346. (I) Pharmacological inhibition of SGK1 blunts NDRG1 phosphorylation in ISO-stimulated brown adipocytes. (J) SGK1 S422D -dependent phosphorylation of RNF34 in HEK293 cells. (K) SGK1 S422D inhibits RNF34-dependent ubiquitination and degradation of GOT1 in HEK293 cells. (L) SGK1 S422D mitigates the inhibitory effect of RNF34 overexpression on GOT1 in brown adipocytes. (M) A schematic describing the role of SGK1 in enhancing GOT1 protein stability by protecting it from ubiquitin/proteasome-dependent degradation. All data are presented as the Mean ± SEM. ** p < 0.01, # p < 0.0001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Gene Expression, Expressing, Activation Assay, Phospho-proteomics, Inhibition, Ubiquitin Proteomics, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: Loss of SGK1 reduces GOT1 protein levels in brown adipocytes without altering its gene expression (A) qPCR analysis of Sgk1 fl/fl and Sgk1 −/− brown adipocytes treated with isoproterenol (ISO) for 4 h. (B) Validation of SGK1 deficiency in Sgk1 −/− brown adipocytes. (C) Loss of SGK1 reduces GOT1 protein levels in ISO-stimulated brown adipocytes. (D) Effects of Sgk1 ablation on mitochondrial respiration in ISO-stimulated brown adipocytes. Left panel: The oxygen consumption rates (OCR) were measured in Sgk1 fl/fl and Sgk1 −/− brown adipocytes after 4 h of ISO stimulation, followed by injections of oligomycin (Oligo), FCCP, and complex III inhibitor antimycin A (AA). Right panel: Total mitochondrial respiration, oligomycin-dependent coupled respiration, and oligomycin-independent leak respiration were determined as described in the Methods section. All data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Gene Expression, Biomarker Discovery

Journal: Cell Reports

Article Title: The hepatic compensatory response to elevated systemic sulfide promotes diabetes

doi: 10.1016/j.celrep.2021.109958

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were; TST, Rabbit, GeneTex, GTX114858, MPST, Rabbit, Abcam, Ab224043, GOT1, Rabbit, Abcam, ab170950, GSTT1, Rabbit, Proteintech, 15838-1-AP, MAT1A, Rabbit, Abcam, ab129176, BHMT Rabbit, Proteintech, 15965-1-AP, CSAD, Rabbit, Abcam, ab91016, PPCS Rabbit, Atlas Antibodies, HPA031361.

Techniques: Recombinant, Protease Inhibitor, Marker, Staining, Sequencing, Modification, Electron Microscopy, Cholesterol Assay, Multiplex sample analysis, Knock-Out, Gene Expression, Software

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Analysis of the effect of NV-161, CB-839, GOT1i, PEPCKi, MB05032, CHC, GSK2837808, CP-91149, and 6AN on cell viability.

Article Snippet: GOT1i , MedChemExpress , HY-122723.

Techniques: Concentration Assay

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: GOT1i , MedChemExpress , HY-122723.

Techniques: Sequencing, Control, Magnetic Beads, Recombinant, Colorimetric Assay, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Cytometry, Mass Spectrometry, Imaging, Spectrophotometry

Journal: bioRxiv

Article Title: Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

doi: 10.1101/459602

Figure Lengend Snippet: ( A ) Relative abundance of cysteine-related metabolites in cell extracts following CDO1 WT or CDO1 Y157F expression in A549 cells. Fresh media was added 4 hr prior to extraction. Metabolites were analyzed by untargeted LC-MS. N.B.-cysteine and GSH were not derivatized with NEM for this analysis. (B) GOT1 mediates the transamination of CSA to produce β-sulfinyl pyruvate, which decomposes to pyruvate and sulfite (SO 3 2- ). Sulfite hydrolyzes (CYS) 2 to produce cysteine-S-sulfate (CYS-SO 3 - ) in a process known as sulfitolysis. (C-D) Time-dependent quantification of the media concentrations of and SO 3 2- ( C ) and CYS-SO 3 - ( D ) concentrations from . (E) Time-dependent quantification of (CYS) 2 and CYS-SO 3 - following the addition of 1mM Na 2 SO 3 to RPMI + 10% FBS in the absence of cells. (F) CYS-SO 3 - production rate of NRF2 KO A549 cells reconstituted with either pLX317 empty (NRF2-) or pLX317-NRF2 (NRF2 +), followed by expression of CDO1 Y157F (Y157F) or CDO1 WT (WT). (G) CYS-SO 3 - production rate of H1975 and H1299 cells expressing either pLX317 empty (-) or pLX317-NRF2 T80K (NRF2 T80K ), followed by expression of CDO1 Y157F (Y157F) or CDO1 WT (WT). (H) CYS-SO 3 - production rate of A549 cells following expression of CDO1 Y157F (Y157F) or CDO1 WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). (I) Intracellular SO 3 2- concentration in the cells from ( H ). ( J-K ) CYS-SO 3 - production rate of H1944 (J) and H322 (K) cells following expression of CDO1 Y157F (Y157F) or CDO1 WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). For A, C-D, F-K, cells were treated with 0.25μg/ml doxycycline for 2 days prior to and during the assay.

Article Snippet: GOT1 knockout cells were generated using the plentiCRISPR-sgGOT1 vector (addgene # 72874), and reconstituted with pMXS-GOT1 (addgene #72872) ( ). pMXS-empty was generated from pMXS-GOT1 by site-directed mutagenesis.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Concentration Assay

Journal: bioRxiv

Article Title: Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

doi: 10.1101/459602

Figure Lengend Snippet: (A) Verification of the stable-isotope labeled CYS-SO 3 - internal standard for absolute quantification. Stable isotope labeled CYS-SO 3 - was synthesized from [ 13 C 3 , 15 N]-cysteine and analyzed by LC-MS/MS based SRM against an authentic standard. RT, retention time. (B) Time-dependent quantification of (CYS) 2 and CYS-SO 3 - following the addition of 1mM CSA to RPMI + 10% FBS in the absence of cells. (C) Western blot analysis of NRF2, CDO1, GOT1, CSAD and β-ACTIN levels following expression of GFP, CDO1 WT , or CDO1 Y157F in KEAP1 WT/WT (WT) and KEAP1 R554Q/R554Q (R554Q) MEFs. A549s expressing CDO1 WT are included for comparison. (D-I) Quantitation of cystine (CYS 2 , D ), cysteine (CYS, E ), glutathione (GSH, F ), cysteine sulfinic acid (CSA, G ), hypotaurine (HTAU, H ) and taurine (TAU, I ) levels in KEAP1 R554Q/R554Q MEFs (red) compared to KEAP1 WT/WT MEFs (black) following expression of CDO1 Y157F (Y157F) or CDO1 WT (WT). (J) Western blot analysis of GOT1, CSAD and HSP90 levels in KEAP1 wild-type and KEAP1 mutant NSCLC cell lines. Mouse liver is included as a positive control for GOT1 and CSAD expression. Cells were fed with fresh RPMI + 10% FBS 4 hours prior to lysate collection. (K) Intracellular SO 3 - concentration of NRF2 KO A549 cells reconstituted with either pLX317 empty (NRF2 -) or pLX317-NRF2 (NRF2 +), followed by re-expression of CDO1 Y157F (Y157F) or CDO1 WT (WT). (L) SO 3 - production rate of cells from ( K ). ( M-O ) Intracellular CSA concentration of A549 ( M ), H1944 ( N ) and H322 ( O ) cells following re-expression of CDO1 Y157F (Y157F) or CDO1 WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT).For C-O , cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay, and fresh media was added 4 hr prior to sample collection.

Article Snippet: GOT1 knockout cells were generated using the plentiCRISPR-sgGOT1 vector (addgene # 72874), and reconstituted with pMXS-GOT1 (addgene #72872) ( ). pMXS-empty was generated from pMXS-GOT1 by site-directed mutagenesis.

Techniques: Labeling, Synthesized, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Quantitation Assay, Mutagenesis, Positive Control, Concentration Assay

Journal: bioRxiv

Article Title: Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

doi: 10.1101/459602

Figure Lengend Snippet: (A) Western blot analysis of GOT1 and β-ACTIN expression in parental (P) A549 and H460 cells, and GOT1 KO clones #1 and #2 for each cell line expressing empty pMXS (GOT1 -) and reconstituted with pMXS-GOT1 (GOT1 +). (B) Contribution of sulfitolysis to (CYS) 2 consumption by CDO1. CDO1-dependent (CYS) 2 consumption, and CSA, SO 3 2- , and CYS-SO 3 - production were determined as cysteine molar equivalents. (C) Analysis of the proliferation of GOT1 KO A549 cells from ( A ) expressing CDO1 Y157F (Y157F) or CDO1 WT (WT). Cells were collected on the indicated days, stained with crystal violet and their absorbance at 600nm determined. (D-E) Quantitation of glutathione (GSH) total ( 12 C + 13 C) levels ( D ) and 13 C-labeling only ( E ) from 13 C-cystine in cells from (A) following expression of CDO1 Y157F (Y157F) or CDO1 WT (WT). 13 C-label is shown in red, while 12 C-label is gray. Cells were labeled for 1 hour. (F) Analysis of Coenzyme A (CoA) labeling from 13 C, 15 N-cystine in cells from ( A ) expressing CDO1 Y157F (Y157F) or CDO1 WT (WT). 13 C, 15 N-label (M+3) is shown in red, while 12 C, 14 N-label is gray. Cells were labeled for 1 hour. (G) Analysis of protein synthesis rates with azidohomoalanine labeling in cells from ( A ) expressing CDO1 Y157F (Y157F) or CDO1 WT (WT). Cells treated with 50μg/mL cyclohexamide (C) were used as a positive control for translation inhibition. For B-G, cells were treated with 0.25μg/ml doxycycline for 2 days prior to and during the assay.

Article Snippet: GOT1 knockout cells were generated using the plentiCRISPR-sgGOT1 vector (addgene # 72874), and reconstituted with pMXS-GOT1 (addgene #72872) ( ). pMXS-empty was generated from pMXS-GOT1 by site-directed mutagenesis.

Techniques: Western Blot, Expressing, Clone Assay, Staining, Quantitation Assay, Labeling, Positive Control, Inhibition

Journal: bioRxiv

Article Title: Cysteine dioxygenase 1 is a metabolic liability for non-small cell lung cancer

doi: 10.1101/459602

Figure Lengend Snippet: (A) The NADPH/NADP+ ratio was assayed following expression of CDO1 Y157F (Y157F) or CDO1 WT (WT) in A549 and H460 GOT1 KO cells expressing empty pMXS (GOT1 -) or reconstituted with pMXS-GOT1 (GOT1 +). (B) A549 and H460 GOT1 KO cells expressing empty pMXS (dashed line, open circle) or reconstituted with pMXS-GOT1 (solid line, solid circle), followed by expression of CDO1 Y157F (gray) or CDO1 WT (red), were treated with 0-25μM cumene hydroperoxide (CuH 2 O 2 ) for 24 hr. Cell numbers were analyzed using crystal violet and normalized to untreated cells. *= GOT1+ CDO1 WT vs. CDO1 Y157F , #= GOT1-CDO1 WT vs. CDO1 Y157F . (C) Analysis of proline (M+5) labeling from L-[U]- 13 C-glutamine in GOT1 KO A549 and H460 cells expressing empty pMXS (Control) or reconstituted with pMXS-GOT1 (GOT1), followed by expression of CDO1 Y157F or CDO1 WT . Proline M+5 abundance was normalized to the abundance of its precursor glutamate M+5, and then CDO1 WT levels were normalized to CDO1 Y157F . Cells were labeled for 1 hour in proline-free media. (D-E) Mass isotopomer analysis of citrate labeling in GOT1 KO A549 ( D ) and H460 ( E ) cells expressing empty pMXS (Control) or reconstituted with pMXS-GOT1 (GOT1) following expression of CDO1 Y157F or CDO1 WT cultured with L-[U]- 13 C-glutamine for 4 hours. (F) Model: CDO1 antagonizes the growth and survival of KEAP1 MUT cells by depleting the intracellular cysteine pool. ( Left ) Elevated intracellular cysteine (CYS) stabilizes CDO1, leading to the production of CSA and SO 3 2- . In turn, SO 3 2- depletes cystine via sulfitolysis to produce CYS-SO 3 - , further limiting cysteine availability and utilization. SO 2 (sulfur dioxide) exists in equilibrium with SO 3 2- and is a gas that may diffuse in and out of cells. The continual reduction of (CYS) 2 to CYS depletes cellular NADPH, limiting its availability for cellular processes. ( Right ) CDO1 silencing promotes the accumulation of intracellular CYS and NADPH, and prevents wasting of CYS as CSA and toxic SO 3 2- . GSR, glutathione reductase. TXNRD1, thioredoxin reductase 1. Fe-S, iron sulfur cluster. For A-E , cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay.

Article Snippet: GOT1 knockout cells were generated using the plentiCRISPR-sgGOT1 vector (addgene # 72874), and reconstituted with pMXS-GOT1 (addgene #72872) ( ). pMXS-empty was generated from pMXS-GOT1 by site-directed mutagenesis.

Techniques: Expressing, Labeling, Cell Culture