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: 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: International Journal of Biological Sciences

Article Title: Pyroptotic Macrophage-Derived Microvesicles Accelerate Formation of Neutrophil Extracellular Traps via GSDMD-N-expressing Mitochondrial Transfer during Sepsis

doi: 10.7150/ijbs.87646

Figure Lengend Snippet: Pyroptotic Macrophage-derived MVs Altered Mitochondrial Homeostasis in Neutrophils. Human peripheral neutrophils were isolated and cultured with PBS, control macrophage-derived MVs, or pyroptotic macrophage-derived MVs. (A, B and C) ΔΨ of human peripheral neutrophils was assessed using JC-1staining. JC-1 monomers (green) and aggregates (red) were assessed using fluorescence microscopy (A) and flow cytometry (B and C). Scale bar, 20 µm. (D and E) MitoSOX of human peripheral neutrophils was assessed using fluorescence microscopy (D) and flow cytometry (E). Scale bar, 50 µm. (F and G) Mitochondrial mass in human peripheral neutrophils was detected using flow cytometry (F) and fluorescence microscopy (G). Scale bar, 20 µm. (H) Representative images showing neutrophil staining of DNA (DAPI, blue), TOM20 (green), and 8-OHG (red) in human peripheral neutrophils following exposure to MVs for 4 h. Results are represented as mean ± SEM.

Article Snippet: Cells were blocked with 1% BSA for 30 min and stained with rabbit anti-histone H3 antibody (Abcam; 1:500); mouse anti-myeloperoxidase antibody (Abcam 1:500); rabbit anti-GSDMD (Abcam; 1:500); rabbit anti-TOM20 antibody (Abcam 1:500); mouse anti-8-OHdG antibody (Novus; 1:200).

Techniques: Derivative Assay, Isolation, Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Staining

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