Journal: Pharmacological research

Article Title: New TIPARP inhibitor rescues mitochondrial function and brain injury in ischemic stroke.

doi: 10.1016/j.phrs.2024.107508

Figure Lengend Snippet: Fig. 9. XG-04-B1 restores mitochondrial homeostasis in dMCAO mice. Mitochondrial homeostasis was analyzed by the protein expression of (A) OPA1, (B) MFN2, (C) DRP1, p-DRP1 Ser637, p-DRP1 Ser616, and β-actin in ischemic cortex of dMCAO mice with XG-04-B1 and edaravone intervention. Data are presented as the means ± SEM. Data were analyzed using one-way ANOVA followed by Holm-Sidak’s post hoc test (A, B, and C). *P<0.05, **P<0.01, ***P<0.001. n=6 mice/group.

Article Snippet: Mouse anti-GFAP antibody (1:1000, G3893, Sigma-Aldrich), mouse anti-PSD95 antibody (1:1000, ab2723, Abcam), rabbit anti-Synaptophysin antibody (1:1000, 17785–1-AP, Proteintech), rabbit anti-TIPARP (1:500, ab84664, abcam), rabbit anti-OPA1 antibody (1:1000, 27733–1-AP, Proteintech), rabbit anti-MFN2 antibody (1:1000, 12186–1-AP, Proteintech), rabbit anti-DRP1 antibody (1:1000, 12957–1-AP, Proteintech), rabbit anti-phospho-DRP1 (Ser637) antibody (1:1000, 6319, Cell Signaling Technology), rabbit anti-phospho-DRP1 (Ser616) antibody (1:1000, 4494, Cell Signaling Technology), rabbit anti-ITGB1 antibody (1:1000, 12594–1-AP, Proteintech), rabbit anti-COXIV antibody (1:5000, 11242–1-AP, Proteintech), rabbit anti-Histone H3 antibody (1:5000, 17168–1-AP, Proteintech), or mouse anti-β-actin antibody (1:3000, 66009–1-lg, Proteintech) were used to detect signals overnight at 4 ◦C.

Techniques: Expressing

Journal: Neuron

Article Title: Long-term potentiation requires a rapid burst of dendritic mitochondrial fission during induction

doi: 10.1016/j.neuron.2018.09.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary: anti-Drp1 (rabbit, D6C7, CST, 1:50), anti-Homer1 (rabbit, Synaptic Systems,1:500), Anti-GluA2 (mouse, Millipore MAB397, 1:100).

Techniques: Virus, Plasmid Preparation, Recombinant, Mutagenesis, Purification, Software

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

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Cardiac‐Specific Deletion of Orai3 Leads to Severe Dilated Cardiomyopathy and Heart Failure in Mice

doi: 10.1161/JAHA.120.019486

Figure Lengend Snippet: A , a and b showing representative transmission electron microscopy pictures of the sarcomere structure of cardiac muscle from 1‐month‐old male mice. Scale bar=500 nm. High magnification images (inset, c‐d) depicting normal Z‐line (arrowhead) and M‐band (red parenthesis) in the control and disorganized Z‐line and shallow M‐band in Orai3 cKO sections. Scale bar=10 nm. B , Cross‐sections through intercalated disc showing large mixed‐type junction (area composita) surrounded by two dense desmosome structures (a and b, black arrows). Representative ultrastructural images of left ventricle from control and Orai3 cKO hearts showing normal (control) and altered (Orai3 cKO ) mitochondrial morphology and density (c and d). C , Graphs summarizing mitochondria number, mitochondrial size, mean cross‐sectional area, and optical. Data are expressed as mean±SEM t‐test, ⁎ P <0.01 for Orai3 cKO vs. control. D and E , Representative photograph and analysis of cardiomyocytes loaded with JC‐1 to measure mitochondrial membrane potential, emissions at 525/585 are visualized in green and red fluorescence, respectively. Scale bar=50 µm. Cells isolated from 3 mice each group, t‐test: * P <0.01. F , Immunoblot and analysis showing that the expression of DRP1 (dynamin‐related protein 1) was increased in Orai3 cKO hearts when compared with the control group. Loading was normalized using total protein (Figure ). DRP1 indicates dynamin‐related protein 1; LV, left ventricle; Orai3 cKO , Orai3 cardiomyocyte‐specific knockout; and TEM, transmission electron microscopy. t‐test: * P <0.01.

Article Snippet: In this study, the following primary antibodies were used for Western blot and immunohistochemical detection: mouse anti‐Orai1 (Santa Cruz, cat. sc‐377281), rabbit anti‐Orai2 (Proteintech, cat. 26766), rabbit anti‐Orai3 (ProSci, cat. 4117), rabbit anti‐TRPC6 (transient receptor potential canonical type 6) (Abcam, cat. ab62641), rabbit anti‐tubulin (Cell Signaling, cat. 2146), rabbit anti‐GAPDH (Cell Signaling, cat. 5174), rabbit anti‐DRP1 (dynamin‐related protein 1) (Invitrogen, cat. pa5‐20176), mouse anti‐SERCA2a (sarco/endoplasmic reticulum Ca 2+ ‐ATPase) (Santa Cruz, cat. sc‐376235), rabbit anti‐NCX1 (Abcam, cat. ab177952), rat anti‐ERTR7 (fibroblast marker, Santa Cruz, cat. sc‐73355, phalloidin (Invitrogen, cat. R415).

Techniques: Transmission Assay, Electron Microscopy, Fluorescence, Isolation, Western Blot, Expressing, Knock-Out

Journal: iScience

Article Title: Circ-CIMIRC inhibition alleviates CIH-induced myocardial damage via FbxL4-mediated ubiquitination of PINK1

doi: 10.1016/j.isci.2024.108982

Figure Lengend Snippet: FbxL4 regulated apoptosis and mitophagy in CIH-treated H9c2 cells H9c2 cells were subjected to CIH treatment, and then transfected with pcDNA-FbxL4, Vector, si-FbxL4 or si-NC. qRT-PCR (A) and western blotting (B) detected the expression of FbxL4 in H9c2 cells. (C) TUNEL staining examined cell apoptosis. (D) IF staining assessed the levels of GFP-LC3 puncta. (E) Western blotting assessed the expression of LC3-I, LC3-II, Mfn2, and Drp1 in H9c2 cells. ∗ p < 0.05, ∗∗ p < 0.01 vs. Control; # p < 0.05, ## p < 0.01 vs. CIH+Vector; $ p < 0.05, $$ p < 0.01 vs. CIH+si-NC.

Article Snippet: The antibodies used in western blotting shown as follows: rabbit anti-FbxL4 (1:1000; bs-13166R; Bioss, Beijing, China), rabbit anti-PINK1 (1:500; 23274-1-AP; Proteintech, Wuhan, China), mouse anti-Parkin (1:500; 39-0900; Thermo Fisher Scientific), rabbit anti-LC3 (1:1000; 14600-1-AP; Proteintech), rabbit anti-cleaved caspase-3 (1:1000; PA5-114687; Thermo Fisher Scientific), rabbit anti-p62 (1:1000; ab240635; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:1000; ab196495; Abcam), rabbit anti-Mfn2 (1:1000; ab124773; Abcam), rabbit anti-Drp1 (1:1000; ab184247; Abcam), rabbit anti-GAPDH (1:10000; ab181602; Abcam), rabbit anti-β-actin (1:1000; ab8227; Abcam), goat anti-mouse IgG (1:5000; ab6789; Abcam), goat anti-rabbit IgG (1:10000; ab6721; Abcam).

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Expressing, TUNEL Assay, Staining

Journal: iScience

Article Title: Circ-CIMIRC inhibition alleviates CIH-induced myocardial damage via FbxL4-mediated ubiquitination of PINK1

doi: 10.1016/j.isci.2024.108982

Figure Lengend Snippet: Circ_CIMIRC repressed PINK1 and Parkin expression, and affected apoptosis and mitophagy of CIH-treated H9c2 cells H9c2 cells were subjected to CIH treatment, and then transfected with pcDNA-circ_CIMIRC, Vector, si-circ_CIMIRC, or si-NC. (A) TUNEL staining examined cell apoptosis. (B–G) Western blotting assessed the expression of FbxL4, PINK1, Parkin, LC3-I, LC3-II, Mfn2, and Drp1 in H9c2 cells. (H) IF staining examined the levels of mitophagy in H9c2 cells. ∗ p < 0.05, ∗∗ p < 0.01 vs. Vector; # p < 0.05, ## p < 0.01 si-NC.

Article Snippet: The antibodies used in western blotting shown as follows: rabbit anti-FbxL4 (1:1000; bs-13166R; Bioss, Beijing, China), rabbit anti-PINK1 (1:500; 23274-1-AP; Proteintech, Wuhan, China), mouse anti-Parkin (1:500; 39-0900; Thermo Fisher Scientific), rabbit anti-LC3 (1:1000; 14600-1-AP; Proteintech), rabbit anti-cleaved caspase-3 (1:1000; PA5-114687; Thermo Fisher Scientific), rabbit anti-p62 (1:1000; ab240635; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:1000; ab196495; Abcam), rabbit anti-Mfn2 (1:1000; ab124773; Abcam), rabbit anti-Drp1 (1:1000; ab184247; Abcam), rabbit anti-GAPDH (1:10000; ab181602; Abcam), rabbit anti-β-actin (1:1000; ab8227; Abcam), goat anti-mouse IgG (1:5000; ab6789; Abcam), goat anti-rabbit IgG (1:10000; ab6721; Abcam).

Techniques: Expressing, Transfection, Plasmid Preparation, TUNEL Assay, Staining, Western Blot

Journal: iScience

Article Title: Circ-CIMIRC inhibition alleviates CIH-induced myocardial damage via FbxL4-mediated ubiquitination of PINK1

doi: 10.1016/j.isci.2024.108982

Figure Lengend Snippet:

Article Snippet: The antibodies used in western blotting shown as follows: rabbit anti-FbxL4 (1:1000; bs-13166R; Bioss, Beijing, China), rabbit anti-PINK1 (1:500; 23274-1-AP; Proteintech, Wuhan, China), mouse anti-Parkin (1:500; 39-0900; Thermo Fisher Scientific), rabbit anti-LC3 (1:1000; 14600-1-AP; Proteintech), rabbit anti-cleaved caspase-3 (1:1000; PA5-114687; Thermo Fisher Scientific), rabbit anti-p62 (1:1000; ab240635; Abcam, Cambridge, MA, USA), rabbit anti-Bcl-2 (1:1000; ab196495; Abcam), rabbit anti-Mfn2 (1:1000; ab124773; Abcam), rabbit anti-Drp1 (1:1000; ab184247; Abcam), rabbit anti-GAPDH (1:10000; ab181602; Abcam), rabbit anti-β-actin (1:1000; ab8227; Abcam), goat anti-mouse IgG (1:5000; ab6789; Abcam), goat anti-rabbit IgG (1:10000; ab6721; Abcam).

Techniques: Recombinant, TUNEL Assay, Apoptosis Assay, Multiple Displacement Amplification, ROS Assay, Immunoprecipitation, Staining, Purification, Software