Journal: Journal of Alzheimer's disease : JAD

Article Title: Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer’s Disease

doi: 10.3233/JAD-170051

Figure Lengend Snippet: Summary of quantitative real-time RT-PCR oligonucleotide primers used in measuring mRNA expression in mitochondrial dynamics, mitochondrial biogenesis, and synaptic genes in in N2a cells treated with Aβ42, Mdivi1+Aβ42, Aβ42+Mdivi1 relative to untreated N2a cells

Article Snippet: An ID fine-band command was used to locate and to scan the bands in each lane and to record the readings. table ft1 table-wrap mode="anchored" t5 caption a7 Marker Primary Antibody – Species and Dilution Purchased from Company, City & State Secondary Antibody, Dilution Purchased from Company, City & State Drp1 Rabbit Polyclonal 1:500 Novus Biological, Littleton, CO Donkey Anti-rabbit HRP 1:10,000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit Polyclonal 1:500 MBL International Corporation-life.

Techniques: Quantitative RT-PCR, Expressing, Sequencing

Journal: Journal of Alzheimer's disease : JAD

Article Title: Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer’s Disease

doi: 10.3233/JAD-170051

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial dynamics, mitochondrial biogenesis, and synaptic proteins in N2a cells treated with Aβ42, Mdivi1+Aβ42, Aβ42+Mdivi1 relative to untreated N2a cells

Article Snippet: An ID fine-band command was used to locate and to scan the bands in each lane and to record the readings. table ft1 table-wrap mode="anchored" t5 caption a7 Marker Primary Antibody – Species and Dilution Purchased from Company, City & State Secondary Antibody, Dilution Purchased from Company, City & State Drp1 Rabbit Polyclonal 1:500 Novus Biological, Littleton, CO Donkey Anti-rabbit HRP 1:10,000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit Polyclonal 1:500 MBL International Corporation-life.

Techniques: Western Blot

Journal: Journal of Alzheimer's disease : JAD

Article Title: Mitochondria-Division Inhibitor 1 Protects Against Amyloid-β induced Mitochondrial Fragmentation and Synaptic Damage in Alzheimer’s Disease

doi: 10.3233/JAD-170051

Figure Lengend Snippet: mRNA fold changes in N2a cells treated with Aβ42 and Mdivi1

Article Snippet: An ID fine-band command was used to locate and to scan the bands in each lane and to record the readings. table ft1 table-wrap mode="anchored" t5 caption a7 Marker Primary Antibody – Species and Dilution Purchased from Company, City & State Secondary Antibody, Dilution Purchased from Company, City & State Drp1 Rabbit Polyclonal 1:500 Novus Biological, Littleton, CO Donkey Anti-rabbit HRP 1:10,000 GE Healthcare Amersham, Piscataway, NJ Fis1 Rabbit Polyclonal 1:500 MBL International Corporation-life.

Techniques:

Journal: Frontiers in oncology

Article Title: Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

doi: 10.3389/fonc.2022.857968

Figure Lengend Snippet: FIGURE 5 | SND1 promotes mitophagy through PGAM5. (A, B) Hep3B cells stably expressing shSND1 (A) or shPGAM5 (B) were treated with 10 mM FCCP for 6 h. Samples were collected for immunoblotting to analyze the expression of SND1, PGAM5, MFN1, TIM23, COX4, p-DRP1S637, and DRP1. Actin served as loading control. LC3 protein levels were detected in Hep3B-shSND1 cells treated with FCCP. (C, D) Hep3B cells stably expressing shSND1 (C) or shPGAM5 (D) were treated with glucose-free medium for 24 h. Samples were collected for immunoblotting to analyze the expression of SND1, PGAM5, MFN1, TIM23, COX4, and p- DRP1S637. Actin served as loading control. LC3 protein levels were detected in Hep3B-shSND1 cells treated with glucose-free medium. (E, F) Hep3B cells stably overexpressing SND1 with further PGAM5 knockdown by shRNAs were treated with FCCP for 6 h (E) or cultured with glucose-free medium for 24 h (F), followed by immunoblotting analysis with antibodies against SND1, PGAM5, MFN1, TIM23, COX4, and p-DRP1S637. Actin served as loading control. (G, H) Hep3B cells stably overexpressing MTS-GFP with further SND1 knockdown (G) or PGAM5 knockdown (H) were cultured with FCCP for 6 h, followed by immunoblotting analysis with antibodies against SND1, PGAM5, MFN1, TIM23, COX4, GFP, and p-DRP1S637. Actin served as loading control. (I) Hep3B cells with endogenous SND1 knockdown were further infected with viruses expressing Flag-EV, Flag-SND1, or Flag-SND1D1-63. These cells were treated with FCCP for 6 h followed by immunoblotting analysis with antibodies against SND1, MFN1, TIM23, COX4, and p-DRP1S637. Actin served as loading control. (J, K) Hep3B cells were infected virus expressing HA-DRP1 together with Flag-PGAM5 plasmids, and the binding activity of PGAM5 and DRP1 under FCCP treatment (J) or glucose deprivation conditions (K) was determined when we further knocked down SND1. Cell lysates were immunoprecipitated with anti-Flag antibody or IgG, followed by immunoblotting analysis with antibodies against HA, Flag, and SND1. Actin served as loading control.

Article Snippet: Primary antibodies against the following proteins were used: SND1 (1:2,000, ab65078, Abcam), PGAM5 (1:1,000, sc-515880, Santa Cruz Biotechnology), TOM20 (1:1,000, 11802-1-AP, Proteintech), TOM70 (1:1,000, 14528-1-AP, Proteintech), COX4 (1:1,000, 11242-1-AP, Proteintech), TIM23 (1:1,000, sc514463, Santa Cruz Biotechnology), MFN1(1:1,000, 13798-1-AP, Proteintech), LC3 (1:1,000, NB100-2220, Novus Biologicals), Phospho-DRP1 (Ser637) (1:1,000, 4867S, CST), DRP1 (1:1,000, 12957-1-AP, Proteintech), Lamin B1 (1:2,000, 12987-1-AP, Proteintech), Tubulin (1:5,000, 66031-1-Ig, Proteintech), bActin (1:2,000, 66009-1-Ig, Proteintech), GAPDH (1:5,000, 60004-1-Ig, Proteintech), TFAM (1:2,000, 22586-1-AP, Proteintech), Flag-M2 (1:2,000, F1804, Sigma-Aldrich), HA (1:1,000, 51064-2-AP, Proteintech), HA-HRP (1:1,000, 2999S, CST), GST (1:5,000, 10000-0-AP, Proteintech), GFP (1:4,000, 50430-2-AP, Proteintech), Calnexin (1:5,000, 10427-2-AP, Proteintech), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) were used.

Techniques: Stable Transfection, Expressing, Western Blot, Control, Knockdown, Cell Culture, Infection, Virus, Binding Assay, Activity Assay, Immunoprecipitation

Journal: Frontiers in oncology

Article Title: Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

doi: 10.3389/fonc.2022.857968

Figure Lengend Snippet: FIGURE 6 | The promotion effect of SND1 on cell proliferation and tumor growth depends on PGAM5. (A, B) Growth curves were measured in SND1- overexpressing Hep3B cells expressing shRNA against PGAM5. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 comparing the indicated samples. (C–E) Equal numbers of Hep3B cells mentioned in panel (A) were injected subcutaneously into the flanks of BALB/c nude mice (n = 5 mice in each group). Tumor sizes were measured every 3 days using calipers (C). Photographs show xenografts (D), and tumor weights (E) were determined at the end of the experiment (Day 35). Data are presented as the mean ± SD, *P < 0.05 comparing the indicated groups. (F) Protein levels of SND1, PGAM5, MFN1, TIM23, COX4, p-DRP1S637, and DRP1 in tumor lysates of panel D were measured by immunoblotting analysis. Actin served as loading control.

Article Snippet: Primary antibodies against the following proteins were used: SND1 (1:2,000, ab65078, Abcam), PGAM5 (1:1,000, sc-515880, Santa Cruz Biotechnology), TOM20 (1:1,000, 11802-1-AP, Proteintech), TOM70 (1:1,000, 14528-1-AP, Proteintech), COX4 (1:1,000, 11242-1-AP, Proteintech), TIM23 (1:1,000, sc514463, Santa Cruz Biotechnology), MFN1(1:1,000, 13798-1-AP, Proteintech), LC3 (1:1,000, NB100-2220, Novus Biologicals), Phospho-DRP1 (Ser637) (1:1,000, 4867S, CST), DRP1 (1:1,000, 12957-1-AP, Proteintech), Lamin B1 (1:2,000, 12987-1-AP, Proteintech), Tubulin (1:5,000, 66031-1-Ig, Proteintech), bActin (1:2,000, 66009-1-Ig, Proteintech), GAPDH (1:5,000, 60004-1-Ig, Proteintech), TFAM (1:2,000, 22586-1-AP, Proteintech), Flag-M2 (1:2,000, F1804, Sigma-Aldrich), HA (1:1,000, 51064-2-AP, Proteintech), HA-HRP (1:1,000, 2999S, CST), GST (1:5,000, 10000-0-AP, Proteintech), GFP (1:4,000, 50430-2-AP, Proteintech), Calnexin (1:5,000, 10427-2-AP, Proteintech), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) were used.

Techniques: Expressing, shRNA, Injection, Western Blot, Control

Journal: Frontiers in oncology

Article Title: Mitochondrion-Localized SND1 Promotes Mitophagy and Liver Cancer Progression Through PGAM5.

doi: 10.3389/fonc.2022.857968

Figure Lengend Snippet: FIGURE 7 | Mitochondrial localization is required for SND1-mediated cell proliferation and tumor growth. (A, B) Growth curves of Hep3B cells with endogenous SND1 knockdown that were expressing Flag-EV, Flag-SND1, or Flag-SND1D1-63 were determined by trypan blue counting. Data are presented as the mean ± SD of three independent experiments. *P < 0.05 comparing the indicated samples. (C–E) Equal numbers of Hep3B cells mentioned in panel (A) were injected subcutaneously into the flanks of BALB/c nude mice (n = 5 mice in each group). Tumor sizes were measured every 3 days using calipers (C). Photographs show xenografts (D), and tumor weights (E) were determined at the end of the experiment (Day 31). Data are presented as the mean ± SD, *P < 0.05 comparing the indicated groups. (F) Protein levels of SND1, MFN1, TIM23, COX4, p-DRP1S637, and DRP1 in tumor lysates of panel (D) were measured by immunoblotting analysis. Actin served as loading control.

Article Snippet: Primary antibodies against the following proteins were used: SND1 (1:2,000, ab65078, Abcam), PGAM5 (1:1,000, sc-515880, Santa Cruz Biotechnology), TOM20 (1:1,000, 11802-1-AP, Proteintech), TOM70 (1:1,000, 14528-1-AP, Proteintech), COX4 (1:1,000, 11242-1-AP, Proteintech), TIM23 (1:1,000, sc514463, Santa Cruz Biotechnology), MFN1(1:1,000, 13798-1-AP, Proteintech), LC3 (1:1,000, NB100-2220, Novus Biologicals), Phospho-DRP1 (Ser637) (1:1,000, 4867S, CST), DRP1 (1:1,000, 12957-1-AP, Proteintech), Lamin B1 (1:2,000, 12987-1-AP, Proteintech), Tubulin (1:5,000, 66031-1-Ig, Proteintech), bActin (1:2,000, 66009-1-Ig, Proteintech), GAPDH (1:5,000, 60004-1-Ig, Proteintech), TFAM (1:2,000, 22586-1-AP, Proteintech), Flag-M2 (1:2,000, F1804, Sigma-Aldrich), HA (1:1,000, 51064-2-AP, Proteintech), HA-HRP (1:1,000, 2999S, CST), GST (1:5,000, 10000-0-AP, Proteintech), GFP (1:4,000, 50430-2-AP, Proteintech), Calnexin (1:5,000, 10427-2-AP, Proteintech), HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) were used.

Techniques: Knockdown, Expressing, Injection, Western Blot, Control

Journal: Nature Communications

Article Title: CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

doi: 10.1038/ncomms13189

Figure Lengend Snippet: ( a ) Increased Drp1 phosphorylation at S616 site (Drp1 S616 ) in the heart after ISO infusion through CaMKII-dependent pathway. N =5, 6, 4 and 4 mice in the groups of Vehicle, ISO, Prop+ISO and KN93+ISO, respectively. ( b ) ISO administration induced Drp1 S616 phosphorylation in cultured adult rat cardiomyocytes. N =6. ( c ) CaMKII blocker, KN-93 (0.5 μM), prevented Drp1 S616 phosphorylation by ISO (100 nM for 12 h). N =4. ( d ) CaMKII WT overexpression induced Drp1 S616 phosphorylation in adult cardiomycytes. N =4. ( e ) Representative immunoblots and quantification of Drp1 proteins in mitochondrial and cytosolic fractions of the heart after ISO infusion. COX IV and β-actin were used as mitochondrial and cytosolic markers, respectively. N =5, 6 and 4 mice in the groups of Vehicle, ISO and KN93+ISO, respectively. ( f ) Immunofluorescent analysis showing increased ‘punctate' Drp1 staining colocalized with mitochondria. Images are representative of 30 cells from three rats in each group. ( g ) Representative immunoblots and quantification of Drp1 S616 or Drp1 S637 phosphorylation in the mitochondrial or cytosolic fractions of the heart after 2-weeks of ISO infusion. N =4. ( h ) ISO treatment (1 μM or 10 μM for 24 h) induced mitochondrial fragmentation in H9C2 cardiac myoblasts. A dominant negative Drp1 mutation (Drp1 K38A) was used as positive control. Form factor (FF; the reciprocal of circularity value) and aspect ratio (AR; major axis/minor axis) were acquired by using ImageJ. Smaller values of AR and FF indicate increased mitochondrial fragmentation. N =17,714, 5,929 and 5,862 mitochondria in the groups of Vehicle, ISO 1 μM and ISO 10 μM, respectively. ( i ) Overexpression of CaMKII WT and CaMKII CA increased mitochondrial fragmentation in H9C2 cells. CaMKII WT potentiated the effects of ISO on mitochondrial morphological change as indicated by significant fragmentation at a low ISO dose (100 nM). N =17,714, 17,692, 9,567 and 17,035 mitochondria in the groups of Vehicle, CaMKII WT, WT+ISO and CaMKII CA, respectively. Data are mean±s.e.m. * P <0.05 versus Vehicle, # P <0.05 versus ISO. The data were analysed using One-way ANOVA followed by Turkey post test.

Article Snippet: After the incubation, the supernatant was used to detect Drp1 phosphorylation by Western blots using anti-P-S616 antibody (1:1,000, Cell Signaling) and rabbit anti-Drp1 antibody (1:2,000, Cell Signaling).

Techniques: Phospho-proteomics, Cell Culture, Over Expression, Western Blot, Staining, Dominant Negative Mutation, Mutagenesis, Positive Control

Journal: Nature Communications

Article Title: CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

doi: 10.1038/ncomms13189

Figure Lengend Snippet: ( a ) Co-immunoprecipitation analysis showing the binding of Drp1 with CaMKII in adult cardiomyocytes. Images are representative of four repeats. ( b ) HA-tagged CaMKII from H9C2 cells were attached to anti-HA magnetic beads and incubated with WT Drp1 or S616A mutation purified from E. coli. (2 μM) in the presence or absence of calmodulin (1 mM) and Ca 2+ (1 mM) for 24 h. The supernatant was used for Western blot to detect Drp1 S616 phosphorylation. Images are representative of three repeats. ( c ) The beads were boiled and samples were used for Western blot using anti-HA (for CaMKII) or anti-Drp1 antibodies for determining the in vitro interaction between CaMKII and Drp1. Images are representative of three repeats.

Article Snippet: After the incubation, the supernatant was used to detect Drp1 phosphorylation by Western blots using anti-P-S616 antibody (1:1,000, Cell Signaling) and rabbit anti-Drp1 antibody (1:2,000, Cell Signaling).

Techniques: Immunoprecipitation, Binding Assay, Magnetic Beads, Incubation, Mutagenesis, Purification, Western Blot, Phospho-proteomics, In Vitro

Journal: Nature Communications

Article Title: CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

doi: 10.1038/ncomms13189

Figure Lengend Snippet: ( a ) Preventing Drp1 S616 phosphorylation by overexpression a phosphorylation null mutation of Drp1 (Drp1 S616A, in which serine at 616 was mutated to alanine) blocked ISO-induced flash frequency in adult cardiomyocytes. N =12, 14, 13 and 10 cells from three rats in the groups of Vehicle, Drp1 S616A, ISO and S616A+ISO, respectively. ( b ) Drp1 S616A also prolonged the time of laser-induced permanent loss of Δ ψ m by chronic ISO treatment (1 μM, 48 h). N =193, 245, 231 and 224 mitochondria from 20 to 24 cells from three rats in the groups of Vehicle, Drp1 S616A, ISO and S616A+ISO, respectively. ( c ) Drp1 S616A rescued myocyte death induced by ISO treatment (10 μM, 48 h). N =1061, 985, 877 and 694 cells from three rats in the groups of Vehicle, Drp1 S616A, ISO and S616A+ISO, respectively. ( d – f ) Drp1 K38A also blocked ISO's effect on flash frequency ( d ), laser-induced permanent loss of Δ ψ m ( e ) and myocyte death ( f ). In d , N =26, 11, 36, 17 cells; in e , N =479, 239, 198 and 196 mitochondria; and in f , N =536, 653, 697 and 819 cells in the groups of Vehicle, Drp1 K38A, ISO and K38A+ISO, respectively. ( g ) Mdivi-1 (50 mg kg −1 d −1 ), a chemical inhibitor of Drp1, efficiently attenuated the increased flash frequency in intact heart by 2 weeks of ISO infusion. N =25, 18 and 27 images from 4 to 6 hearts in the groups of Vehicle, ISO and Mdivi-1+ISO, respectively. ( h ) Mdivi-1 prevented ISO-induced cardiac hypertrophy. N =7–8 mice. Data in a – h are mean±s.e.m. * P <0.05 versus Vehicle group, # P <0.05 versus ISO group. ( i ) Representative images and summarized data showing Drp1 S616 phosphorylation and total Drp1 levels in the ventricular samples of heart failure patients. N =4 for each group. * P <0.05 versus None-failing control group. Data are mean±s.e.m. In a – i , the data were analysed using One-way ANOVA followed by Turkey post test.

Article Snippet: After the incubation, the supernatant was used to detect Drp1 phosphorylation by Western blots using anti-P-S616 antibody (1:1,000, Cell Signaling) and rabbit anti-Drp1 antibody (1:2,000, Cell Signaling).

Techniques: Phospho-proteomics, Over Expression, Mutagenesis, Control

Journal: Nature Communications

Article Title: CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

doi: 10.1038/ncomms13189

Figure Lengend Snippet: Sustained ISO treatment activates CaMKII pathway, a downstream kinase of β1-AR, and subsequently increases the phosphorylation of Drp1 at S616 (Drp1 S616 ), which activates Drp1. After translocating to the outer membrane of mitochondria, the phosphorylated Drp1 triggers fission and mPTP openings, which are recorded by mitochondrial flash events. Finally, chronic activation of this pathway leads to mitochondrial and myocyte dysfunction. Abolishing CaMKII activity (CaMKII DN, KN93 or AIP), inhibiting Drp1 activity (Drp1 K38A or Mdivi-1), preventing Drp1 S616 phosphorylation (Drp1 S616A), or blocking mPTP openings (CsA or CypD KO) efficiently prevented myocyte damage and cardiac hypertrophy during chronic β1-AR stimulation.

Article Snippet: After the incubation, the supernatant was used to detect Drp1 phosphorylation by Western blots using anti-P-S616 antibody (1:1,000, Cell Signaling) and rabbit anti-Drp1 antibody (1:2,000, Cell Signaling).

Techniques: Phospho-proteomics, Membrane, Activation Assay, Activity Assay, Blocking Assay