Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 1. Mitochondria-nucleus contacts. Mesenchymal stem cells (MSCs) were cultured in low-glucose (LG), adipogenic (AD) and osteogenic (OS) media for 3, 7, and 21 days, as indicated. (a) Representative 3D images of mitochondrial morphology, as detected by anti-TOM20 Ab immunofluorescence, and contact sites identified by creating the isosurface of the colocalization channel between nuclear staining (Hoechst) and TOM20. Magnification 40x. Scale bar 10 µm (in zoom panel, scale bar 5 µm). (b) Quantification of mitochondrial total volume, number, and total volume and number of contacts. Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments. Data are shown as the mean ± SD. ANOVA, * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to the LG condition at the same time point.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Staining, Derivative Assay

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 2. Mitochondria in MSC differentiation processes. (a) Representative immunoblot and (b) quantification of HSP60, TOM20, VDAC, and TIM23 protein levels normalized to GAPDH levels. (c) XF phenograms representing the metabolic switching of MSCs during differentiation, as detected using an Extracellular Flux Analyzer (Seahorse Bioscience). (d,e) Oxygen consumption rate (OCR) measurements and relative derived parameters after the addition of oligomycin [1 µM], 1 µM carbonylcyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP) [1 µM], and antimycin A [2 µM] + rotenone [2 µM] in MSCs cultured in low-glucose (LG), adipogenic (AD), and osteogenic (OS) media for (d) 7 and (e) 21 days. Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA, * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with respect to the LG condition at the same time point.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Western Blot, Derivative Assay, Cell Culture

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 4. Inhibition of the citrate transport system impaired mitochondrial behavior. MSCs were cultured in low-glucose (LG), adipogenic (AD), and osteogenic (OS) media; where indicated, BTC [5 mM] and iCTP [500 µM] were added to inhibit the transport of citrate produced in mitochondria to the cytosol. (a) Representative images and (b) analysis of mitochondrial total volume and number detected by the anti-TOM20 Ab and the total volume and number of mitochondrion-nucleus contact sites. Magnification 40x. Scale bar 10 µm (in zoom panel, scale bar 5 µm). Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA-test. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Inhibition, Cell Culture, Produced, Derivative Assay

Journal: Cells

Article Title: Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

doi: 10.3390/cells9041034

Figure Lengend Snippet: Figure 5. Citrate from mitochondria increases the level of α-ketoglutarate to promote osteogenesis. (a) Citrate can be converted to acetyl-CoA by citrate lyase (inhibited by BMS) or to α-ketoglutarate (αKG), two metabolites that can mediate mitochondrion-nucleus communication. (b,c) Analysis of relative mRNA levels of osteogenic markers (RUNX2, RANKL, osteocalcin (OC), osteopontin (OPN), osterix (OSX), and alkaline phosphatase (ALP)) in MSCs cultured in low-glucose (LG) and osteogenic media (OS); where indicated, BMS [1 mM], BTC [5 mM], and αKG [1 mM] were added for 21 days. (d) Representative images and analysis of mitochondrial total volume and number detected by the anti-TOM20 Ab and the total volume and number of mitochondrion-nucleus contact sites in MSCs cultured in LG and osteogenic media (OS); where indicated, BTC [5 mM] and αKG [1 mM] were added for 21 days. (e) Representative images and quantification of H3K9me3 in MSCs cultured in LG and OS media; where indicated, BTC [5 mM] and αKG [1 mM] were added for 21 days. Magnification 40x. Scale bar 10 µm (in zoom 5 µm). Data are derived from ≥45 acquired cells/condition from ≥3 independent experiments and are shown as the mean ± SD. ANOVA-test. * p < 0.5, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used for immunofluorescence images: TOM20 [sc-11415] (1:100) from Santa Cruz Biotechnology, H3 [14269] (1:100), H3K9ac [9649] (1:100), and H3K9me3 [13969] (1:100) from Cell Signaling (Danvers, MA, USA).

Techniques: Cell Culture, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation * ^♦

doi: 10.1074/jbc.M115.671446

Figure Lengend Snippet: Parkin mutants that have a defect of binding with phosphorylated ubiquitin. A, S65E phosphomimetic Ash-Parkin (referred to as WT) and its mutants were expressed with S65D phosphomimetic hAG-Ub (referred to as WT) and its I44A mutant in HeLa cells as the indicated pair in B. Foci of the hAG-Ub (S65D) were observed using confocal microscopy. Scale bars, 20 μm. B, quantification of foci formation in A. The percentages of cells forming foci are shown. The error bars represent ± S.D. from three independent replicates. Over 100 cells were counted in each of three replicate wells. C, the indicated YFP-human Parkin mutants were transiently expressed in HeLa cells. Cells were treated with valinomycin for 2.5 h and subjected to immunostaining with anti-TOMM20 antibody. Scale bars, 20 μm. D, quantification of Parkin translocation in C. The percentages of cells having YFP-Parkin on mitochondria are shown. The error bars represent ± S.D. from three independent replicates. Over 100 cells were counted in each of three replicate wells.

Article Snippet: Rabbit anti-TOMM20 (Santa Cruz Biotechnology, sc-11415) antibody was used for immunostaining.

Techniques: Binding Assay, Mutagenesis, Confocal Microscopy, Immunostaining, Translocation Assay

Journal: Endocrinology

Article Title: Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis

doi: 10.1210/endocr/bqad124

Figure Lengend Snippet: Characteristics of antibodies used for Western blotting and microscopy

Article Snippet: Antibody name Dilution ratio Species specificity Source Supplier (distributor) Catalog No RRID PLIN 2 1:1000 Bovine Guinea pig pAB Fitzgerald Industries International (Acton) 20R-AP002 AB_1282475 PLIN 3 1:1000 Bovine Guinea pig pAB Fitzgerald Industries International 20R-TP001 AB_1289040 STAR 1:10000 Mouse Rabbit pAB Abcam ab96637 AB_10678397 CYP11A1 1:1000 Mouse Rabbit mAB Cell Signaling 14217 AB_2631970 HSD3B 1:1000 a /1:200 b Bovine Mouse mAB Thermo Fisher MA1-46438 AB_2120102 VIM 1:1000 Bovine Rabbit pAB Abcam ab137321 AB_2921312 HSL 1:1000 Mouse Rabbit pAB Cell Signaling 4107 AB_2296900 COX IV 1:1000 Bovine Rabbit mAB Cell Signaling 4850 AB_2085424 HSP47 1:1000 Bovine Mouse mAB Enzo Life Sciences Inc SPA-470 AB_2185189 TOM70 1:1000 Mouse Mouse pAB Abcam ab89624 AB_2043080 CNX 1:200 Mouse Rabbit mAB Cell Signaling 2679 AB_2228381 TOM20 1:200 c Mouse Rabbit mAB Cell Signaling 42406S AB_2687663 TUBB 1:5000 Bovine Mouse mAB Sigma Life Science T4026 AB_477577 ACTB 1:5000 Bovine Mouse mAB Sigma Life Science A5441 AB_476744 Lipi-Blue 1 μM Dojindo Molecular LD01 BODIPY 493/503 10 μM Thermo Fisher D3922 MitoTracker Red FM 200nM Thermo Fisher M22425 HRP-linked 1:10000 Anti-guinea pig Jackson ImmunoResearch 106-035-003 AB_2337402 HRP-linked 1:10000 Anti-rabbit Jackson ImmunoResearch 111-035-003 AB_2313567 HRP-linked 1:10000 Anti-mouse Jackson ImmunoResearch 115-035-205 AB_10015289 DyLight 405 1:500 Anti-mouse Jackson ImmunoResearch 115-475-166 AB_2338786 Alexa Fluor 488 1:500 Anti-mouse Invitrogen A32723 AB_2633275 Alexa Fluor 594 1:500 Anti-rabbit Invitrogen A-11012 AB_2534079 Alexa Fluor 647 1:500 Anti-biotin BioLegend 405237 AB_2941953 Open in a separate window Abbreviations: ACTB, beta-actin (loading control); CNX, calnexin; COX IV, cytochrome c oxidase subunit 4; CYP11A1, cholesterol side-chain cleavage enzyme; HRP, horseradish peroxidase; HSD3B, 3 beta-hydroxysteroid dehydrogenase); HSL, hormone sensitive lipase; HSP47, heat shock protein 47; PLIN2, perilipin 2; PLIN3, perilipin 3; STAR, steroidogenic acute regulatory protein; TOM20, mitochondrial import receptor subunit 20; TOM70, mitochondrial import receptor subunit 70; TUBB, beta-tubulin (loading control); VIM, vimentin. a Dilution used for Western blotting. b Dilution used for confocal microscopy. c Biotinylated antibody.

Techniques: Western Blot

Journal: Endocrinology

Article Title: Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis

doi: 10.1210/endocr/bqad124

Figure Lengend Snippet: Subcellular localization of 3beta-hydroxysteroid dehydrogenase (HSD3B) in steroidogenic small luteal cells. Populations of small luteal cells were enriched from bovine luteal tissue and visualized using confocal microscopy. A, Representative confocal micrograph of HSD3B (red) and luteal lipid droplets (green), calnexin (endoplasmic reticulum; green), or TOM20 (mitochondria; green). White boxes represent an area of 10× enlargement. White dashed lines represent the area of intensity overlap. B, Representative quantitative analysis of intensity overlap. Arrows represent spatial location of lipid droplet. TOM20 (streptavidin, Alexa Fluor 647); calnexin (Alexa Fluor 594); lipid droplet (BODIPY493/503), and HSD3B (DyLight 405). Micron bar represents 20 and 2 µm, respectively.

Article Snippet: Antibody name Dilution ratio Species specificity Source Supplier (distributor) Catalog No RRID PLIN 2 1:1000 Bovine Guinea pig pAB Fitzgerald Industries International (Acton) 20R-AP002 AB_1282475 PLIN 3 1:1000 Bovine Guinea pig pAB Fitzgerald Industries International 20R-TP001 AB_1289040 STAR 1:10000 Mouse Rabbit pAB Abcam ab96637 AB_10678397 CYP11A1 1:1000 Mouse Rabbit mAB Cell Signaling 14217 AB_2631970 HSD3B 1:1000 a /1:200 b Bovine Mouse mAB Thermo Fisher MA1-46438 AB_2120102 VIM 1:1000 Bovine Rabbit pAB Abcam ab137321 AB_2921312 HSL 1:1000 Mouse Rabbit pAB Cell Signaling 4107 AB_2296900 COX IV 1:1000 Bovine Rabbit mAB Cell Signaling 4850 AB_2085424 HSP47 1:1000 Bovine Mouse mAB Enzo Life Sciences Inc SPA-470 AB_2185189 TOM70 1:1000 Mouse Mouse pAB Abcam ab89624 AB_2043080 CNX 1:200 Mouse Rabbit mAB Cell Signaling 2679 AB_2228381 TOM20 1:200 c Mouse Rabbit mAB Cell Signaling 42406S AB_2687663 TUBB 1:5000 Bovine Mouse mAB Sigma Life Science T4026 AB_477577 ACTB 1:5000 Bovine Mouse mAB Sigma Life Science A5441 AB_476744 Lipi-Blue 1 μM Dojindo Molecular LD01 BODIPY 493/503 10 μM Thermo Fisher D3922 MitoTracker Red FM 200nM Thermo Fisher M22425 HRP-linked 1:10000 Anti-guinea pig Jackson ImmunoResearch 106-035-003 AB_2337402 HRP-linked 1:10000 Anti-rabbit Jackson ImmunoResearch 111-035-003 AB_2313567 HRP-linked 1:10000 Anti-mouse Jackson ImmunoResearch 115-035-205 AB_10015289 DyLight 405 1:500 Anti-mouse Jackson ImmunoResearch 115-475-166 AB_2338786 Alexa Fluor 488 1:500 Anti-mouse Invitrogen A32723 AB_2633275 Alexa Fluor 594 1:500 Anti-rabbit Invitrogen A-11012 AB_2534079 Alexa Fluor 647 1:500 Anti-biotin BioLegend 405237 AB_2941953 Open in a separate window Abbreviations: ACTB, beta-actin (loading control); CNX, calnexin; COX IV, cytochrome c oxidase subunit 4; CYP11A1, cholesterol side-chain cleavage enzyme; HRP, horseradish peroxidase; HSD3B, 3 beta-hydroxysteroid dehydrogenase); HSL, hormone sensitive lipase; HSP47, heat shock protein 47; PLIN2, perilipin 2; PLIN3, perilipin 3; STAR, steroidogenic acute regulatory protein; TOM20, mitochondrial import receptor subunit 20; TOM70, mitochondrial import receptor subunit 70; TUBB, beta-tubulin (loading control); VIM, vimentin. a Dilution used for Western blotting. b Dilution used for confocal microscopy. c Biotinylated antibody.

Techniques: Confocal Microscopy

Journal: Endocrinology

Article Title: Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis

doi: 10.1210/endocr/bqad124

Figure Lengend Snippet: Subcellular localization of 3beta-hydroxysteroid dehydrogenase (HSD3B) in steroidogenic large luteal cells. Enriched populations of large luteal cells from bovine luteal tissue were visualized using confocal microscopy. A, Representative confocal micrograph of HSD3B (red) and luteal lipid droplets (green), calnexin (endoplasmic reticulum; green), or TOM20 (mitochondria; green). White boxes represent an area of 10× enlargement. White dashed lines represent the area of intensity overlap. B, Representative quantitative analysis of intensity overlap. Arrows represent spatial location of lipid droplet. TOM20 (streptavidin, Alexa Fluor 647); calnexin (Alexa Fluor 594); lipid droplet (BODIPY493/503), and HSD3B (DyLight 405). Micron bar represents 20 and 2 µm, respectively.

Article Snippet: Antibody name Dilution ratio Species specificity Source Supplier (distributor) Catalog No RRID PLIN 2 1:1000 Bovine Guinea pig pAB Fitzgerald Industries International (Acton) 20R-AP002 AB_1282475 PLIN 3 1:1000 Bovine Guinea pig pAB Fitzgerald Industries International 20R-TP001 AB_1289040 STAR 1:10000 Mouse Rabbit pAB Abcam ab96637 AB_10678397 CYP11A1 1:1000 Mouse Rabbit mAB Cell Signaling 14217 AB_2631970 HSD3B 1:1000 a /1:200 b Bovine Mouse mAB Thermo Fisher MA1-46438 AB_2120102 VIM 1:1000 Bovine Rabbit pAB Abcam ab137321 AB_2921312 HSL 1:1000 Mouse Rabbit pAB Cell Signaling 4107 AB_2296900 COX IV 1:1000 Bovine Rabbit mAB Cell Signaling 4850 AB_2085424 HSP47 1:1000 Bovine Mouse mAB Enzo Life Sciences Inc SPA-470 AB_2185189 TOM70 1:1000 Mouse Mouse pAB Abcam ab89624 AB_2043080 CNX 1:200 Mouse Rabbit mAB Cell Signaling 2679 AB_2228381 TOM20 1:200 c Mouse Rabbit mAB Cell Signaling 42406S AB_2687663 TUBB 1:5000 Bovine Mouse mAB Sigma Life Science T4026 AB_477577 ACTB 1:5000 Bovine Mouse mAB Sigma Life Science A5441 AB_476744 Lipi-Blue 1 μM Dojindo Molecular LD01 BODIPY 493/503 10 μM Thermo Fisher D3922 MitoTracker Red FM 200nM Thermo Fisher M22425 HRP-linked 1:10000 Anti-guinea pig Jackson ImmunoResearch 106-035-003 AB_2337402 HRP-linked 1:10000 Anti-rabbit Jackson ImmunoResearch 111-035-003 AB_2313567 HRP-linked 1:10000 Anti-mouse Jackson ImmunoResearch 115-035-205 AB_10015289 DyLight 405 1:500 Anti-mouse Jackson ImmunoResearch 115-475-166 AB_2338786 Alexa Fluor 488 1:500 Anti-mouse Invitrogen A32723 AB_2633275 Alexa Fluor 594 1:500 Anti-rabbit Invitrogen A-11012 AB_2534079 Alexa Fluor 647 1:500 Anti-biotin BioLegend 405237 AB_2941953 Open in a separate window Abbreviations: ACTB, beta-actin (loading control); CNX, calnexin; COX IV, cytochrome c oxidase subunit 4; CYP11A1, cholesterol side-chain cleavage enzyme; HRP, horseradish peroxidase; HSD3B, 3 beta-hydroxysteroid dehydrogenase); HSL, hormone sensitive lipase; HSP47, heat shock protein 47; PLIN2, perilipin 2; PLIN3, perilipin 3; STAR, steroidogenic acute regulatory protein; TOM20, mitochondrial import receptor subunit 20; TOM70, mitochondrial import receptor subunit 70; TUBB, beta-tubulin (loading control); VIM, vimentin. a Dilution used for Western blotting. b Dilution used for confocal microscopy. c Biotinylated antibody.

Techniques: Confocal Microscopy

Journal: Biomedicines

Article Title: Isoproterenol-Induced Permeability Transition Pore-Related Dysfunction of Heart Mitochondria Is Attenuated by Astaxanthin

doi: 10.3390/biomedicines8100437

Figure Lengend Snippet: Effects of the administration of AST and injection of ISO on the concentrations of mitochondrial respiratory chain complexes in rat heart mitochondria under mitochondrial permeability transition pore (mPTP) opening. Protein samples were extracted from control and threshold [Ca 2+ ] samples and subjected to Western blotting. Changes in mitochondrial complexes were detected using a Total OXPHOS Rodent WB Antibody Cocktail. The immunodetection of Tom20 was used as a loading control. ( a ) Immunostaining with the OXPHOS antibody cocktail and Tom20; ( b – f ) quantification of immunostaining by computer-assisted densitometry (subunit α of complex V (CV-ATP5A-55 kDa), cytochrome b–c1 complex subunit 2 of complex III (CIII-UQCRC2-48 kDa), mitochondrially encoded cytochrome c oxidase I subunit of complex IV (CIV-MTCO1-40 kDa), succinate dehydrogenase [ubiquinone] iron–sulfur subunit of complex II (CII-SDHB-30 kDa), and dehydrogenase [ubiquinone] 1 β subcomplex subunit 8 of complex I (CI-NDUFB820 kDa), respectively). The data are presented as the means ± SDs of three independent experiments. * p < 0.05 indicates a significant difference in the protein level relative to the control (group 1). # p < 0.05 compared with RHM isolated from ISO-injected rats (group 3). The statistical significance of the differences between the pairs of mean values was evaluated using an ANOVA type 2 (Student–Newman–Keuls) test.

Article Snippet: The Tom20 antibody (1:1000 dilution; Cell Signaling, Danvers, MA, USA) was used as a loading control.

Techniques: Injection, Permeability, Control, Western Blot, Immunodetection, Immunostaining, Isolation

Journal: Biomedicines

Article Title: Isoproterenol-Induced Permeability Transition Pore-Related Dysfunction of Heart Mitochondria Is Attenuated by Astaxanthin

doi: 10.3390/biomedicines8100437

Figure Lengend Snippet: Effects of the administration of AST and injection of ISO on the content of the mitochondrial protein adenine nucleotide translocase (ANT) and the voltage-dependent anion channel (VDAC) in rat heart mitochondria isolated from rats from each group under mPTP opening. ( a ) Western blots stained with corresponding antibodies under control and threshold [Ca 2+ ] conditions; ( b ) diagrams quantitatively reflecting changes in the ANT content in absolute units normalized to Tom20; ( c ) diagrams quantitatively reflecting changes in the level of VDAC in absolute units normalized to Tom20. The data are presented as the means ± SDs of three independent experiments. * p < 0.05 indicates a significant difference in the protein level relative to the control (group 1). # p < 0.05 compared to RHM isolated from ISO-injected rats (group 3). The statistical significance of the differences between the pairs of the mean values was evaluated using an ANOVA type 2 (Student–Newman–Keuls) test.

Article Snippet: The Tom20 antibody (1:1000 dilution; Cell Signaling, Danvers, MA, USA) was used as a loading control.

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

Journal: Biomedicines

Article Title: Isoproterenol-Induced Permeability Transition Pore-Related Dysfunction of Heart Mitochondria Is Attenuated by Astaxanthin

doi: 10.3390/biomedicines8100437

Figure Lengend Snippet: Effects of the administration of AST and injection of ISO on the content of the mitochondrial protein 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), subunit b, and subunit c in rat heart mitochondria isolated from rats of each group under mPTP opening. ( a ) Western blots stained with the corresponding antibodies under control and threshold [Ca 2+ ] conditions; ( b ) diagrams quantitatively reflecting changes in the CNPase content in absolute units normalized to Tom20; ( c ) diagrams quantitatively reflecting changes in the level of subunit b in absolute units normalized to Tom20; ( d ) diagrams quantitatively reflecting changes in the level of subunit c in absolute units normalized to Tom20. The data are presented as the means ± SDs of three independent experiments. * p < 0.05 indicates a significant difference in the protein level relative to the control (group 1). # p < 0.05 compared with RHM isolated from ISO-injected rats (group 3). The statistical significance of the differences between the pairs of mean values was evaluated using an ANOVA type 2 (Student–Newman–Keuls) test.

Article Snippet: The Tom20 antibody (1:1000 dilution; Cell Signaling, Danvers, MA, USA) was used as a loading control.

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

Journal: Nature Communications

Article Title: Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis

doi: 10.1038/s41467-022-30490-6

Figure Lengend Snippet: a – c RT-qPCR analysis of si CTRL and si ATG5 LEC treated with sodium acetate (AC, 20 mM, 48 h) or vehicle. mRNA expression of Prox1, VEGFR3, and LYVE1 (relative to HPRT). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 and *** p < 0.001 vs si CTRL, ## p < 0.01 and ### p < 0.001 vs si ATG5. d Representative blots for the indicated proteins in si CTRL or si ATG5 LEC treated with AC or vehicle. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05,*** p < 0.001. e Representative blots for indicated proteins in si CTRL and si ATG5 LEC treated with palmitate (PAL, 500 nM, 48 h) or BSA. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. f Representative immunofluorescent images of si CTRL and si ATG5 treated with AC or vehicle and stained for mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. g Quantification of mitochondrial fragmentation index (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05 vs si CTRL, # p < 0.05 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 12 cells analyzed per condition per independent experiment. h Quantification of lipid droplet number per cell. Mean ± SD, N = 4 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, * p < 0.05 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 29 cells analyzed per condition per independent experiment. i Representative super resolution AiryScan images of si CTRL and si ATG5, si CTRL + si DRP1, and si ATG5 + si DRP1 LEC stained for the mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. j Quantification of mitochondrial index of fragmentation (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, **** p < 0.0001 vs si CTRL, #### p < 0.0001 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 35 cells analyzed per condition per independent experiment. k Quantification of lipid droplet number per cell. Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 47 cells analyzed per condition per independent experiment. l – o RT-qPCR analysis in si CTRL, si ATG5, si CTRL + si DRP1 and si ATG5 + si DRP1 LEC. mRNA expression of PROX1, VEGFR3, LYVE1, and CPT1A (relative to HPRT). Mean ± SD, N = 3 ( l , n , o ) and N = 4 ( m ) biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ** p < 0.01 vs si CTRL and p = ns vs si ATG5.

Article Snippet: Primary antibodies used were rabbit anti-ATG5 (12994S, CST), rabbit anti-ATG7 (1:1000, 8558S, CST), rabbit anti-LC3 (1:1000, 3868S, CST), rabbit anti-GAPDH (1:5000, 2118S, CST), rabbit anti-p62 (1:1000, p0067, Millipore), rabbit anti-β-actin (1:5000, A5441, Sigma-Aldrich), goat anti-LYVE1 (1:1000, AF2089, R&D systems), rabbit anti-PROX1 (1:1000, 11067-2, Proteintech), rabbit anti-VEGFR3 (1:1000, ab154079, Abcam), mouse anti-NR2F2 (1:1000, ab41859, Abcam), rabbit anti-CPT1 (1:1000, D3B3, CST) antibody, rabbit anti-CPT2 (1:1000, ab18114, Abcam), rabbit anti-acetyl histone H3 (lysine 9) antibody (1:1000, 9671, CST), rabbit anti-pan-acetyl histone H3 antibody (1:1000, 39139, Active Motif)), mouse anti-CD36 antibody (1:1000; ab17044, Abcam), mouse anti-ULK1 antibody (1:1000; ab56344, Abcam), rabbit anti-TOMM20 (1:1000; BD612278, BD Biosciences), mouse anti-OPA1 (1:1000; 612607, BD Biosciences), mouse anti-ENOS (1:1000; 610297, BD biosciences), rabbit anti-phospho DRP1 (Ser616) (1: 1000; 3455S, CST), mouse anti-DRP1 (1:1000; 611113, BD Biosciences), rabbit anti-H3K4me antibody (1:1000; ab8580, Abcam), rabbit anti-H3K27 ac (1:1000; 8173T, CST) and rabbit anti-FABP4 antibody (1:1000; 2120S, CST).

Techniques: Quantitative RT-PCR, Expressing, Staining