Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: A , B MDA-MB-231 and MCF-7 cells were treated with or without flubendazole (0.5 μM) for 24 h. A fluorescence microscope evaluated the intensity of Calcein AM. Representative images and quantification of Calcein AM were shown. Scale bar, 5 µm. C , D Flow cytometric analysis and quantification of mitochondrial membrane potential changes in MDA-MB-231 and MCF-7 cells treated with or without flubendazole (0.5 μM) for 24 h. E Mitochondria were stained with MitoTracker TM Deep Red FM probes for 30 min and observed with a confocal microscope. Representative images of mitochondrial morphology were shown. Scale bar, 5 µm. F RT-qPCR analysis of mitochondrial DNA copies in MDA-MB-231 and MCF-7 cells. G , H Mitochondria were stained with MitoSOX TM Red FM for 30 min, and mitochondrial ROS accumulation was analyzed by flow cytometry. I ATP content measurement in MDA-MB-231 and MCF-7 cells treated with or without flubendazole (0.5 μM) for 24 h. J Immunoblotting analysis of VDAC1, SOD2, COX IV, TOM20 expression in MDA-MB-231 and MCF-7 cells treated with the indicated concentration of flubendazole for 24 h. β-actin was used as the loading control. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Fluorescence, Microscopy, Membrane, Staining, Quantitative RT-PCR, Flow Cytometry, Western Blot, Expressing, Concentration Assay, Control

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: A MDA-MB-231 and MCF-7 cells were treated with or without flubendazole (0.5 μM) for 24 h. The images were captured with a transmission electron microscope. Scale bar, 500 nm. B , C The autophagosomes are labeled by LC3 (green fluorescence) protein and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 5 µm. D Immunoblotting of PINK1, Parkin, p-Parkin ser65 and LC3 in MDA-MB-231 and MCF-7 cells treated with the indicated concentrations of flubendazole for 24 h. β-actin was used as the loading control. E Immunoblotting of Parkin in the cytosolic and mitochondrial fractions of MDA-MB-231 and MCF-7 cells treated with or without Flubendazole (0.5 μM) for 24 h. β-actin (cytoplasmic fraction) and VDAC1 (mitochondrial fraction) were used as the loading controls. F , G Colocalization of PINK1 (green fluorescence) protein and Parkin (red fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized PINK1 and Parkin was quantified. Scale bar, 10 µm. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Transmission Assay, Microscopy, Labeling, Fluorescence, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: A Immunoblotting of DRP1, p-DRP1 ser616 and Mitofusin-2 in MDA-MB-231 and MCF-7 cells treated with the indicated concentrations of flubendazole for 24 h. β-actin was used as the loading control. B DRP1 mRNA expression in MDA-MB-231 and MCF-7 cells was analyzed by RT-qPCR. C , D MDA-MB-231 and MCF-7 cells were transfected with negative-control or DRP1 shRNA for 24 h, respectively. After treatment with or without flubendazole (0.5 μM) for 24 h. The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 5 µm. E , F Colocalization of PINK1 (green fluorescence) protein and Parkin (red fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized PINK1 and Parkin was quantified. Scale bar, 10 µm. G Immunoblotting of Parkin, p-Parkin ser65 and PINK1 expression. β-actin was measured as the loading control. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Transfection, Negative Control, shRNA, Labeling, Fluorescence

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: MDA-MB-231 and MCF-7 cells were transfected with EVA1A siRNA or negative-control for 24 h, respectively, followed by treatment with or without flubendazole (0.5 μM). A Immunoblotting of EVA1A, DRP1, p-DRP1 ser616 , Parkin, p-Parkin ser65 and PINK1 expression. β-actin was measured as the loading control. B , C The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 10 µm. D , E Colocalization of Parkin (red fluorescence) protein and TOM20 (green fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized Parkin and TOM20 was quantified. Scale bar, 10 µm. F ATP content measurement. G , H Flow cytometric analysis and quantification of mitochondrial membrane potential changes. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Transfection, Negative Control, Western Blot, Expressing, Control, Labeling, Fluorescence, Membrane

Journal: Cell Death & Disease

Article Title: Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer

doi: 10.1038/s41419-022-04823-8

Figure Lengend Snippet: MDA-MB-231 and MCF-7 cells were co-transfected with DRP1 shRNA and Flag-EVA1A or vehicle control respectively for 48 h. A Immunoblotting of DRP1, EVA1A, p62, Parkin, p-Parkin ser65 , PINK1, and LC3 expression. β-actin was measured as the loading control. B , C The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 10 µm. D , E Colocalization of Parkin (red fluorescence) protein and TOM20 (green fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized Parkin and TOM20 was quantified. Scale bar, 10 µm. Data represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical significance compared with respective control groups (all P -values were obtained by one-way ANOVA).

Article Snippet: Antibodies used in this study were as follow: DRP1 (1:1000, 184247, Abcam, USA), p-DRP1 Ser616 (1:1000, 3455, CST, USA), Mitofusin-2 (1:1000, 9482, CST, USA), VDAC1 (1:1000, 55259, Proteintech, USA), SOD2 (1:1000, 13194, CST, USA), COX IV (1:1000, 4844, CST, USA), TOM20 (1:1000, 42406, CST, USA), PINK1 (1:500, 23274-1-AP, Proteintech), Parkin (1:500, 14060-1-AP, Proteintech, USA), p-Parkin ser65 (1:1000, 36866, CST, USA), LC3 (1:1000, 51520, Abcam, USA), p62 (1:1000, 8025, CST, USA), MMP-2 (1:1000, 87809, CST, USA), E-cadherin (1:1000, 14472, CST, USA), EVA1A (1:1000, 216043, Abcam, USA), β-actin (1:1000, 66009-1-Ig, Proteintech, USA), MitoTracker TM Deep Red FM (M22426, Invitrogen, USA).

Techniques: Transfection, shRNA, Control, Western Blot, Expressing, Labeling, Fluorescence

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