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: Autophagy

Article Title: Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.

doi: 10.1080/15548627.2022.2132686

Figure Lengend Snippet: Figure 1. Effect of FK506, a PPP3/calcineurin inhibitor, on mitophagy of INS-1 insulinoma cells after treatment with mitochondrial stressors. (A) INS-1 cells transfected with pMito-Keima plasmid were incubated with rotenone (Rot) or oligomycin+antimycin A (O/A) for 18 h in the presence or absence of FK506 pretreatment. Fluorescent microscopy was performed at excitation wavelengths of 440 and 590 nm to visualize fluorescence from mitochondrial Keima and that from acidic Mito- Keima delivered to lysosome, thus from mitophagy, respectively (left). The number of acidic Mito-Keima puncta per cell (middle) and Mito-Keima red:green ratio representing mitophagy (right) were calculated. Scale bar: 5 μm. n = 30 (Veh, vehicle; Rot, rotenone; O/A, oligomycin+antimycin A). (B) INS-1 cells transfected with mRFP-LC3 were treated for 24 h and stained with Ab to TOMM20, a mitochondrial outer membrane protein, to visualize autophagosomes colocalized with TOMM20, thus the occurrence of mitophagy (left). The number of RFP puncta colocalized with TOMM20 was counted (right). Scale bar: 5 μm. n = 20. (C) INS-1 cells transfected with pMito-Keima were cultured without mitochondrial stressors in the presence or absence of FK506 for 72 h. Fluorescent microscopy was performed as in (A) (left). The number of acidic Mito-Keima puncta per cell (middle) and Mito-Keima red:green ratio representing mitophagy (right) were calculated. Scale bar: 10 μm. n = 30. (D) INS-1 cells transfected with pMito-Keima were incubated in hypoxic chamber (1% O2) for 24 h with or without FK506. Fluorescence microscopy was performed as in (A) (left). The number of acidic Mito-Keima puncta per cell (right upper) and Mito-Keima red:green ratio representing mitophagy (right lower) were calculated. Scale bar: 5 μm. n = 20. (E and F) After O/A treatment of INS-1 cells for 1 (E) or 24 h (F) with or without FK506 pretreatment for 1 h, cells were stained with MitoTracker Green to visualize mitochondria (left). Average length of mitochondria was measured to estimate mitochondrial fission (right). Scale bar: 10 μm. n = 8. Cells in the rectangles were magnified. All data in this figure are the means ± SEM from more than three independent experiments. **P < 0.01, ***P < 0.001 by one- way ANOVA with Tukey’s test (A,B,D,E,F) or two-tailed t-test (C). ns: not significant.

Article Snippet: To visualize mitophagy in another way, cells transfected with the mRFPLC3 construct were treated with mitochondrial stressors and then incubated with anti-TOMM20 Ab (Cell Signaling Technology, 42406S; 1:200).

Techniques: Transfection, Plasmid Preparation, Incubation, Microscopy, Fluorescence, Staining, Membrane, Cell Culture, Two Tailed Test

Journal: Autophagy

Article Title: Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.

doi: 10.1080/15548627.2022.2132686

Figure Lengend Snippet: Figure 2. Effect of FK506 on TFEB nuclear translocation. (A) After treatment of TFEB-GFP- transfected INS-1 cells with rotenone or O/A for 4 h with or without FK506 pretreatment for 1 h, fluorescent microscopy was conducted (left). TFEB nucleus:cytosol fluorescence ratio was determined using ImageJ software (right). Scale bar: 50 μm. n = 50. (B) After rotenone or O/A treatment of TFE3-GFP-transfected INS-1 cells with or without FK506 pretreatment as in (A), fluorescent microscopy was conducted (left). TFE3 nucleus:cytosol fluorescence ratio was determined (right). Scale bar: 50 μm. n = 50. (C) After treatment of INS-1 cells with rotenone or O/A with or without pretreatment with FK506, cells were fractionated by centrifugation. Cytoplasmic and nuclear fractions were subjected to immunoblot analysis using the indicated Abs. Densitometric values after ImageJ analysis of the band intensity normalized to that of GAPDH (middle) or LMNA (lamin A) band (right) were compared between groups. Representative immunoblots are presented (left). n = 3. (D) After transfection of INS-1 cells with TFEB-GFP or constitutively active TFEB∆N30-GFP together with mRFP-LC3, cells were treated with rotenone or O/A for 4 h with or without FK506 pretreatment for 1 h. After immunostaining with anti-TOMM20 Ab, mRFP-LC3 puncta colocalized with TOMM20 were visualized by confocal microscopy as in Figure 1B (left). The number of mRFP-LC3 puncta colocalized with TOMM20 was counted (right). Scale bar: 5 μm. n = 20. All data in this figure are the means ± SEM from more than three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s test.

Article Snippet: To visualize mitophagy in another way, cells transfected with the mRFPLC3 construct were treated with mitochondrial stressors and then incubated with anti-TOMM20 Ab (Cell Signaling Technology, 42406S; 1:200).

Techniques: Translocation Assay, Transfection, Microscopy, Fluorescence, Software, Centrifugation, Western Blot, Immunostaining, Confocal Microscopy

Journal: Autophagy

Article Title: Impaired TFEB activation and mitophagy as a cause of PPP3/calcineurin inhibitor-induced pancreatic β-cell dysfunction.

doi: 10.1080/15548627.2022.2132686

Figure Lengend Snippet: Figure 5. Effect of FK506 on glucose profile and β-cell function in vivo. (A) C57BL/6 mice were treated with daily administration of 10 mg/kg FK506 for 8 weeks with or without combined administration of 50 mg/kg MSL-7 treatment three times a week. Non-fasting blood glucose (left) and body weight (right) were monitored. n = 5. (B) After treatment of C57BL/6 for 8 weeks as in (A), intraperitoneal GTT was performed (left). Area under the curve (AUC) was calculated (right). n = 11. (C) In mice treated with FK506 as in (A), insulinogenic index was calculated as described in the Materials and methods. n = 9–15. (D) Mitochondrial COX activity in pancreatic islets of mice treated as in (B) was determined as described in the Materials and methods, and expressed as the mean pixel intensity per islet area (right). Representative DAB images are presented (left). Scale bar: 50 μm. n = 5. (E) GFP-RFP-LC3-transgenic mice were treated with daily administration of 10 mg/kg FK506 for 8 weeks with or without combined administration of 50 mg/kg MSL-7 treatment three times a week for 8 weeks. The number of RFP puncta colocalized with TOMM20 in pancreatic islets was counted (middle). Representative fluorescence images of RFP puncta colocalized with TOMM20 are presented (left). The number of red puncta representing autolysosome or autophagic flux and that of yellow puncta representing autophagosome was counted (right). Arrows indicate RFP puncta colocalized with TOMM20. Scale bar: 5 μm. n = 8–10. (F and G) In pancreatic sections of the mice of (E), colocalization of LAMP2 spot, a lysosomal marker, with TOMM20 was estimated by Pearson’s correlation analysis (F). Representative fluorescence images are presented (G). Arrows indicate LAMP2 spots colocalized with TOMM20. Scale bar: 10 μm. n = 8. (H and I) %mitophagy level in pancreatic islets of FK506-injected Mito-Keima-transgenic mice with or without combined administration of MSL-7 was calculated as described in the Materials and method (H). Representative Mito-Keima red fluorescence images in pancreatic islets identified by insulin immunofluorescence are presented (I). Scale bar: 10 (boxed areas) or 20 µm. n = 6–7. Cells in the rectangles were magnified. All data in this figure are the means ± SEM from more than three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s test (B-F) or two- way ANOVA with Bonferroni’s test (A,B). ns: not significant.

Article Snippet: To visualize mitophagy in another way, cells transfected with the mRFPLC3 construct were treated with mitochondrial stressors and then incubated with anti-TOMM20 Ab (Cell Signaling Technology, 42406S; 1:200).

Techniques: Cell Function Assay, In Vivo, Activity Assay, Transgenic Assay, Fluorescence, Marker, Injection, Immunofluorescence