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