Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a, Schematic representation of the principle of lipid labeling using NBD-CoA through lipid remodeling. b-e, Confocal imaging analysis of newly remodeled and NBD-labeled phospholipids in live C2C12 cells. C2C12 cells were incubated with NBD-palmitoyl-CoA alone ( b ), or NBD-palmitoyl-CoA and LPA ( c ), LPG ( d ), or MLCL ( e ). Mitochondria were stained with MitoTracker-Red. Arrows highlight the co-localization of NBD-PL with mitochondria.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Labeling, Imaging, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a, Time-lapse confocal imaging analysis of NBD labeling and mitochondrial localization of PA in C2C12 cells. Arrowheads highlight the co-localization of NBD-PA with mitochondria. Arrows highlight the co-localization of NBD-PA with mitochondria. b, Time-lapse confocal imaging analysis of NBD labeling and the ER localization of PA in C2C12 cells. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPA. Images were taken at the indicated times after incubation. Mitochondria were stained with Mitotracker Red. ER was visualized by transfecting cells with DsRed-ER5.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a, Time-lapse confocal imaging analysis of NBD labeling and mitochondrial localization of CL in C2C12 cells. Arrow-heads highlight the co-localization of NBD-CL with mitochondria. Arrows highlight the co-localization of NBD-CL with mitochondria. b, Time-lapse confocal imaging analysis of NBD labeling and the ER localization of CL in C2C12 cells. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and MLCL. Images were taken at the indicated times after incubation. Mitochondria were stained with Mitotracker Red. ER was visualized by transfecting cells with DsRed-ER5.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a, Time-lapse confocal imaging analysis of NBD labeling and mitochondrial localization of PC in C2C12 cells. Arrows highlight the co-localization of NBD-PC with mitochondria. b, Time-lapse confocal imaging analysis of NBD labeling and the ER localization of PC in C2C12 cells. Arrows highlight the co-localization of NBD-PC with the ER. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPC. Images were taken at the indicated times after incubation. Mitochondria were stained with Mitotracker Red. ER was visualized by transfecting cells with DsRed-ER5.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a-c, TLC analysis of newly remodeled PA ( a ), PG ( b ) or PC ( c ) in C2C12 cells. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA alone or with LPA, LPG or LPC for 15 min, respectively. d, TLC analysis of newly remodeled TAG in COS-7 cells. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and MAG or DAG for 15 min. The total lipids were extracted and developed by TLC as described in Methods .

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Incubation

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: Primary mouse embryonic fibroblasts ( a ) and primary mouse hepatocytes ( b ) were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA alone or NBD-palmitoyl-CoA plus LPA or LPG for 15 min. Mitochondria were stained with Mitotracker Red.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a-c, Confocal imaging analysis of the labeling and mitochondrial transport of PA in C2C12-vector control ( a ), PRELID1 KO ( b ) and TRIAP1 KO ( c ) cells. Arrows highlight the co-localization of NBD-PA with mitochondria. d-e, Confocal imaging analysis of the labeling and mitochondrial transport of PG in C2C12-vector control ( d ), PRELID1 KO ( e ) and TRIAP1 KO ( f ) cells. Arrows highlight the NBD-PG outside of mitochondria. g, Pearson’s correlation coefficient of mitochondria and NBD-PA in vector, PRELID1 KO and TRIAP1 KO cells. n=21-23 cells/group. h-i, Pearson’s correlation coefficient of mitochondria and NBD-PG in vector and PRELID1 KO ( h ) or TRIAP1 KO cells ( i ). n=30 cells/group. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPA ( a-c ) or LPG ( d-f ) for 15 min. Mitochondria were stained with MitoTracker-Red. Data are represented as mean ± SD, **p<0.01, ***p<0.001, ns, no significance by student’s t-test.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Plasmid Preparation, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a-c, Time-lapse confocal imaging analysis of the labeling and mitochondrial transport of PA in C2C12-vector control ( a ), PRELID1 KO ( b ) and TRIAP1 KO ( c ) cells. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPA. Images were taken at the indicated times after incubation. Mitochondria were stained with Mitotracker Red.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Plasmid Preparation, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a-c, Confocal imaging analysis of the labeling and mitochondrial transport of PC in C2C12-vector control ( a ), PRELID1 KO ( b ) and TRIAP1 KO ( c ) cells. Arrows highlight the co-localization of NBD-PC with mitochondria. ( d-e ) Pearson’s correlation coefficient of mitochondria and NBD-PC in vector and PRELID1 KO ( d ) or TRIAP1 KO cells ( e ). n=30 cells/group. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPC for 15 min. Mitochondria were stained with Mitotracker Red. Data are represented as mean ± SD, ns, no significance by student’s t-test.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Plasmid Preparation, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a-b, Confocal imaging analysis of the labeling and mitochondrial transport of PC in C2C12-vector control ( a ) and StARD7 KO ( b ) cells. c, Pearson’s correlation coefficient of mitochondria and NBD-PC in vector and StARD7 KO cells. N=25-29 cells/group d-e, Confocal imaging analysis of the labeling and mitochondrial transport of PG in C2C12-vector control ( d ) and StARD7 KO ( e ) cells. f, Pearson’s correlation coefficient of mitochondria and NBD-PG in vector and StARD7 KO cells. N=30 cells/group. g-h, Confocal imaging analysis of the labeling and mitochondrial transport of PA in C2C12-vector control ( g ) and StARD7 KO ( h ) cells. i, Pearson’s correlation coefficient of mitochondria and NBD-PA in vector and StARD7 KO cells. N=30 cells/group. Cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPC ( a-b ), LPG ( d-e ), or LPA ( g-h ) for 15 min. Mitochondria were stained with MitoTracker-Red. Data are represented as mean ± SD, ***p<0.001, ns, no significance by student’s t-test.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Plasmid Preparation, Incubation, Staining

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a-b, Confocal imaging analysis of the labeling and mitochondrial transport of cholesterol ester. C2C12 cells were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA alone ( a ) or NBD-palmitoyl-CoA plus cholesterol ( b ) for 15 min. Mitochondria were stained with MitoTracker-Red. Arrows highlight the co-localization of NBD-lipid with mitochondria. c, Confocal imaging analysis of cholesterol esterification inhibition by treatment with avasimibe. C2C12 cells were pre-treated with avasimibe for 24 hr. Cells were then starved in KRPH buffer for 1 hr and then incubated with NBD-palmitoyl-CoA and cholesterol. d, Pearson’s correlation coefficient of mitochondria and NBD in the presence or absence of avasimibe. N=30 cells/group. e - f, Measurement of the total cell and mitochondrial cholesterol esters (CE, e ) and free cholesterol (CHOL, f ) in C2C12 cells. Cells pretreated with or without avasimibe were starved in KRPH buffer for 1 hr, and then incubated with palmitoyl-CoA alone (-CHOL) or palmitoyl-CoA plus cholesterol (+CHOL, or +CHOL + Avasimibe) for 30 min. N=3/group. g-h, Confocal imaging analysis of the labeling and mitochondrial transport of cholesterol ester in the presence ( h ) or absence ( g ) of etomoxir. C2C12 cells were pre-treated with or without etomoxir for 4 hrs, and then incubated with palmitoyl-CoA plus cholesterol for 15 min. Data are represented as mean ± SD, ***p<0.001 by Students t-test ( d ) or one-way ANOVA ( e-f ).

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Incubation, Staining, Inhibition

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a, Confocal imaging analysis of COS-7 cells incubated with NBD-palmitoyl-CoA and 2-Oleoylglycerol (MAG). Arrows indicate the newly synthesized lipid droplets. b, Confocal imaging analysis of COS-7 cells incubated with NBD-palmitoyl-CoA and 1,2-Dioleoyl-sn-glycerol (DAG). Arrows indicate the co-localization of NBD-TAG with lipid droplets. COS-7 cells were starved in KRPH buffer containing Bodipy 650/665 to label lipid droplets for 1 hr before incubating with NBD-CoA plus MAG or DAG. ER was visualized by transfecting cells with DsRed-ER5.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Incubation, Synthesized

Journal: bioRxiv

Article Title: De Novo Lipid Labeling for Comprehensive Analysis of Subcellular Distribution and Trafficking in Live Cells

doi: 10.1101/2020.10.16.342683

Figure Lengend Snippet: a, Confocal imaging analysis of the labeling and mitochondrial localization of PE. C2C12 cells were starved in KRPH buffer for 1 hr and then incubated with NBD-palmitoyl-CoA and LPE for 15 min. Mitochondria were stained with MitoTracker-Red. Arrows indicate the NBD-PE punctas. b, Confocal imaging analysis of the co-localization of newly remodeled NBD-PE with autophagosomes. COS-7 cells transfected with mRFP-LC3 were starved in KRPH buffer for 1 hr, and then incubated with NBD-palmitoyl-CoA and LPE for 15 min.

Article Snippet: For lipid labeling, cells were first nutrient starved in a standard Krebs Ringer Phosphate HEPES buffer (KRPH, 140 mM NaCl, 2 mM Na 2 HPO 4 , 4 mM KCl, 1 mM MgCl 2 , 1.5 mM CaCl 2 , 10 mM HEPES, pH7.4) for 1 hr and then incubated with 16:0 NBD-CoA (1 μM, Avanti Polar Lipids, Cat# 810705) and lysophospholipids (20-100 μM, Avanti Polar Lipids) in KRPH buffer for 10-15 min. For visualizing of mitochondria, cells were either transfected with Mito-BFP, or stained with Mitotracker Red before nutrient starvation in complete medium (CM) for 15 min. For visualizing the endoplasmic reticulum (ER), cells were either transfected with DsRed2-ER5, or stained with ERtracker Red before nutrient starvation in CM for 15 min.

Techniques: Imaging, Labeling, Incubation, Staining, Transfection

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: ACOT activities of reconstituted ABCD1. ( A ) Thioesterase activity of reconstituted ABCD1. Proteoliposomes containing ABCD1 (1.84 μg) or negative control liposomes containing non-specific protein (1.62 μg) were incubated with 2 μM NBD-C16-CoA for 10, 20, and 30 min at 37 °C. The “no incubation (0 min)” was run as the reaction was stopped before the addition of NBD-C16-CoA. Each reaction was subjected to TLC to separate NBD-C16-CoA and NBD-C16. ( B ) The ACOT activity was determined after the amount of NBD-C16 was quantified using the image analysis software Image J, and the ACOT activity of ABCD1 (filled circle) and non-specific protein (open circle) are shown. Error bars indicate the standard error (n = 3). ( C ) Acylation of ABCD1 with NBD-C16-CoA. ABCD1-liposomes were incubated with NBD-C16-CoA or NBD-C16 for 30 min at 37 °C and then subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow head indicates ABCD1.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Negative Control, Incubation, Software, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Amino acid residues responsible for the ACOT activity of ABCD1 ( A ) Proteoliposomes containing ABCD1 (2.70 μg) were incubated with NBD-C16-CoA in the presence or absence of each compound (1 mM) for 30 min at 37 °C. Aliquots were subjected to TLC as shown in Fig. A. Relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J (upper graph). ACOT activity without any reagents (0.27 nmol/min/mg) has been normalized to 1. The aliquots were also subjected to SDS-PAGE and the acylation of ABCD1 with NBD-C16-CoA was analyzed (lower panel). Error bars indicate the standard error (n = 3). The arrow head indicates ABCD1. PMSF: phenylmethylsulfonyl fluoride, DFP: diisopropylfluorophosphate, BNPP: bis-(4-nitrophenyl)phosphate, p CMB: p -chloromercuribenzoic acid, DEPC: diethyl pyrocarbonate. ( B ) ACOT activity was measured in the presence of various concentrations of p CMB for 30 min at 37 °C. Aliquots were subjected to TLC as shown in Fig. B. Relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J. ACOT activity without p CMB (0.29 nmol/min/mg) has been normalized to 1. ( C ) Stability of covalent linkage between ABCD1 and NBD-C16. ABCD1-liposomes were incubated with NBD-C16-CoA for 30 min at 37 °C and then subjected to SDS-PAGE. The gels were fixed and then divided. Each gel was incubated with 1 M Tris–HCl pH 7.0, 1 M Hydroxylamine pH 7.0, 0.1 N KOH or 0.1 N HCl for 18 h at room temperature, respectively. The gels were stained with CBB after the detection of NBD fluorescence. The arrow head indicates ABCD1.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Incubation, Software, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Substrate specificity of ABCD1-catalyzed ACOT activity. ( A ) Proteoliposomes containing ABCD1 (3.04 μg) were incubated with NBD-C16-CoA or NBD-C16-3′-dephosphoCoA (NBD-C16-dePiCoA) for 30 min at 37 °C. The aliquots were subjected to TLC. ( B ) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow head indicates ABCD1. ( C ) Proteoliposomes containing ABCD1 (3.16 μg) were incubated with NBD-C16-CoA or NBD-C6-CoA for 30 min at 37 °C. The aliquots were subjected to TLC. ( D ) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow head indicates ABCD1.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Incubation, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Relationship between ATPase activity and the ACOT activity of ABCD1 ( A ) Effect of ATP on ABCD1-catalyzed ACOT activity. ABCD1-liposomes were incubated with NBD-C16-CoA in the presence of various concentration of ATP for 30 min at 37 °C. The aliquots were subjected to TLC as shown in Fig. . The relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J. Error bars indicate the standard error (n = 3). ( B ) His-ABCD1(a.a.1–431) and His-ABCD1(K513A) were prepared using the same procedure as the purification of His-ABCD1 and then reconstituted into liposomes. Proteoliposomes containing ABCD1wild type, ABCD1(K513A) or ABCD1(1–431) (2.68 μg, 3.21 μg or 3.71 μg, respectively) were incubated with NBD-C16-CoA for 30 min at 37 °C. The aliquots were subjected to TLC to separate hydrolyzed NBD-C16 and NBD-C16-CoA. ( C ) Relative ACOT activities of ABCD1(a.a.1–431) and ABCD1(K513A) were evaluated. The intensities of NBD-C16 were normalized by the signal intensities of each of the proteins obtained by immunoblot analysis using the image analysis software Image J. Error bars indicate the standard error (n = 3). Wild type activity (0.33 nmol/min/mg) has been normalized to 1. ( D ) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow heads indicate the wild type, a.a.1–431 or K513A.

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Activity Assay, Incubation, Concentration Assay, Software, Purification, Western Blot, SDS Page, Staining, Fluorescence

Journal: Scientific Reports

Article Title: Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

doi: 10.1038/s41598-021-81949-3

Figure Lengend Snippet: Transport of the NBD-C16 derived from NBD-C16-CoA into ABCD1- liposomes. ( A ) Proteoliposomes containing ABCD1 (4.81 μg) or non-specific protein were incubated with NBD-C16-CoA at 37 °C for the indicated periods. After incubation, the remaining NBD-C16-CoA in the outside portion of the liposomes was quenched with sodium dithionite. In some cases, the liposomes were treated with sodium dithionite after the addition of 0.01% Triton X-100. Subsequently, ABCD1-liposomes were precipitated by centrifugation and then resuspended with 80% acetone. NBD-C16 and NBD-C16-CoA were separated by TLC. ( B ) Transport of NBD-C16 into ABCD1-liposomes was tested under various conditions. ABCD1-liposomes were incubated with NBD-C16-CoA in the presence of p CMB (1 mM) or palmitoyl-CoA (20 μM), or in the absence of ATP. NBD-C16-3′-dephosphoCoA (NBD-C16-dePiCoA) and NBD-C6-CoA were also examined as the substrate. Relative NBD-C16 transport activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J (right graph). Error bars indicate the standard error (n = 3). (C) NBD-C16 transport activity for ABCD1(K513A) was tested by the same procedure as used for the wild type. The relative transport activity ABCD1(K513A) was evaluated as follows. The intensities of NBD-C16 were normalized by the signal intensities of the wild type and K513A obtained by immunoblot analysis using the image analysis software Image J. Error bars indicate the standard error (n = 3).

Article Snippet: NBD-C16-CoA was incubated with Nuclease P1 (FUJIFILM Wako, Osaka, Japan) in 50 mM Tris–HCl pH 8.0 and 1 mM DTT at 37 °C for 3 h, then subjected to TLC.

Techniques: Derivative Assay, Incubation, Centrifugation, Software, Activity Assay, Western Blot