To observe the effect of Mdivi-1 on septic liver injury and STING signaling activation in KCs, Mdivi-1 (S7162; Selleck, Houston, TX) was prepared by dissolving in DMSO and was intraperitoneally administrated at 20 mg/kg or equal volume of Vehicle DMSO at 2 h prior to CLP. To observe the role of STING signaling in septic liver injury, DMXAA (S1537; Selleck, Houston, TX), the agonist of STING, was prepared by dissolving in DMSO and was intraperitoneally administrated at 10 mg/kg at 2 h prior to CLP in wild-type (WT) mice. To observe whether the protective effects of Mdivi-1 on the liver injury through inhibiting STING signaling, Mdivi-1 and DMXAA were administrated in CLP mice at the same time.
Mdivi 1
Manufactured by Selleck Chemicals
Sourced in United States, China
Mdivi-1 is a small molecule that functions as an inhibitor of the mitochondrial fission protein Drp1. It is commonly used in scientific research to study the role of mitochondrial dynamics in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Dmxaa, by Selleck Chemicals (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mitotempo, by Merck Group (1 mentions)
Ha ub, by GenePharma (1 mentions)
Streptomycin, by Beyotime (1 mentions)
Penicillin, by Beyotime (1 mentions)
Aml12 cells, by American Type Culture Collection (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Lipofectamine 3000, by GenePharma (1 mentions)
Nocodazole, by Selleck Chemicals (1 mentions)
Cvt 313, by Selleck Chemicals (1 mentions)
Mdivi 1, by Beyotime (1 mentions)
Mitotracker red, by Beyotime (1 mentions)
27 protocols using mdivi 1
1
Mdivi-1 Modulates Septic Liver Injury
2
Hypoxia/Reoxygenation Stress Response in AML12 Cells
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AML12 cells (ATCC, Manassas, VA, USA) were cultured in DMEM: F12 medium (Gibco, Carlsbad, CA, USA) supplemented with FBS (Gibco), ITTS, and dexamethasone. AML12 cells were exposed to hypoxia/reoxygenation (H/R) as described previously [29 (link)]. In brief, AML12 cells were cultured at 1% O2 for 12 h, and then at 21% O2 conditions for the indicated time points. Plasmids or small interfering RNAs (siRNAs) were transfected to AML12 cells using lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 48 h before H/R. The specific siRNA sequences (GenePharma, Suzhou, China) were listed in Table 1 . After transfection, AML12 cells were incubated with 100 μM MitoTEMPO (Sigma, St Louis, Missouri, USA) or 10 μM Mdivi-1 (Selleck, Shanghai, China) for 3 h, followed by H/R. In addition, 20 μM MG132 (Selleck, USA) was administered for 3 h or 100 μM cycloheximide (CHX, Sigma) was applied for 0, 1, 2, and 4 h to AML12 cells after USP15 siRNA transfection.
HEK293 cells were grown in DMEM (Gibco) medium containing FBS (Gibco), penicillin and streptomycin (Beyotime, Hangzhou, China). Relevant plasmids including Myc-p66Shc, Flag-USP15 WT, Flag-USP15 C269A, and HA-Ub (GenePharma) were transfected into HEK293 cells using lipofectamine 3000 for 24–72 h, followed by a Co-IP assay.
HEK293 cells were grown in DMEM (Gibco) medium containing FBS (Gibco), penicillin and streptomycin (Beyotime, Hangzhou, China). Relevant plasmids including Myc-p66Shc, Flag-USP15 WT, Flag-USP15 C269A, and HA-Ub (GenePharma) were transfected into HEK293 cells using lipofectamine 3000 for 24–72 h, followed by a Co-IP assay.
3
Selective CDK2 Inhibition and Mitochondrial Dynamics
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Two μmol/L CVT‐313 (Selleck), an effective ATP‐competitive selective CDK2 inhibitor, was incubated for 12 h and used for UMUC3 and T24. Mdivi‐1 (Selleck), a mitochondrial division inhibitor 1, is a selective transmembrane inhibitor of mitochondrial division that inhibits DRP1 and dynamin I (Dnm1). Five μmol/L Mdivi‐1 was incubated with the cell cultures for 24 h. Mito‐Tracker Red (Beyotime) was used at a concentration of 200 μmol/L for 15 min to stain the mitochondria. Nocodazole (Selleck) was applied at a dosage of 100 nmol/L for 12 h to induce the synchronization of cells. Then, the synchronized cells were released in a fresh medium.
4
Assessing Mitochondrial Dynamics and Autophagy
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For all the studies, (R)-9b treatment was given in 1% serum condition at concentration 1 μM until specifically indicated for time intervals as per experiments. WT and Mut-ATP5F1A cells were cultured in media with 1% serum for 12 h and processed for immunofluorescence-based detection of MitoTracker (Thermo fisher scientific, M7512) staining. In these experiments Oligomycin (Oligo; Sigma, 579–13-5) treatment (0.5 μM) for 3 h was used as control for depletion of MitoTracker intensity. WT and Mut-ATP5F1A cells were cultured in media with 1% serum for 72 h for immunofluorescence-based detection of LC3B-II, LysoTracker (Invitrogen, L12492) staining. For experiments involving TMRE (Abcam, ab113852)-based immunofluorescence or flow cytometry, cells were treated with Oligo (0.5 μM) for 1 h or FCCP (Sigma, 370–86-5) (0.5 μM) for 1 h prior to harvesting cells. For experiments involving 3-MA (Santa Cruz Biotechnology, sc-205,596; 50 μM) and Mdivi-1 (Selleckchem, S7162; 50 μM) treatments, WT and Mut-ATP5F1A cells were cultured in 1% serum for 12 h followed by 48, 72 or 96 h of (R)-9b treatment, as per experiments. sodium orthovanadate (Thermo Fisher Scientific, S454-50; 1 μM) and sodium fluoride (Sigma, S6521; 1 μM) were added to culture media for inhibition of protease for 3 h prior to termination of respective experiment.
5
Baicalein Inhibits Mitochondrial Dynamics
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Baicalein (≥ 99%, Yousi Scientific Co., Ltd, Shanghai, China) was dissolved in DMSO at concentration of 200 mM and stored at -20 ℃. Mdivi-1, an inhibitor of Drp1, was purchased from Selleck (Huston, TX, USA). 3-Methyladenine (3-MA), an inhibitor of autophagosomes, and Bafilomycin A1 (Baf-A1), an inhibitor of H+-ATPase, were purchased from Selleck. Antibodies against PARP (#9542), Drp1 (#5391), AMPKα (#5831), p-AMPKα (Thr172) (#2535), LC3 (#12741), Bak (#6947) and β-actin (#3700) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies against Caspase 3 (#19677-1-AP), Caspase 9 (#10380-1-AP), Bcl2 (#12789-1-AP), Bcl-xl (#10783-1-AP), Bax (#50599-1-AP), Cytochrome c (#10993-1-AP), Aif (#17984-1-AP), Cox IV (#11242-1-AP), Fis1 (#10956-1-AP), Opa1 (#27733-1-AP), Mfn1 (#13798-1-AP), Ndufs1 (#12444-1-AP), Sdha (#14865-1-AP), Uqcrc1 (#21705-1-AP), Atp5a1 (#14676-1-AP), p62 (#18420-1-AP), and Beclin1 (#11306-1-AP) were obtained from Proteintech (Wuhan, China). Antibody against p-Drp1 (Ser616) (#12749) was obtained from Signalway Antibody (College Park, MD, USA). Secondary goat anti-rabbit or rabbit anti-mouse antibodies were purchased from Proteintech. Fluorescent-labeled antibody Annexin V-FITC, Annexin V-APC, PI, 7-AAD and 10 × binding buffer were obtained from BD (Franklin Lakes, New Jersey, USA).
6
Mitochondrial Dynamics Regulation in Toxicity
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Isoniazid and SB203580 (SB) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Mdivi-1, an inhibitor of DRP1, was purchased from Selleck Chemicals (Houston, TX, United States). Tetramethyl rhodamine methyl ester (TMRM), MitoTracker Deep Red FM, Hoechst 33342 and Lipofectamine RNAiMAX were obtained from Invitrogen (Grand Island, NY, United States). p38 MAPK-siRNA, a silencer negative control siRNA, and antibodies against p38 MAPK, phospho-p38 MAPK, NRF1, COX IV, cytochrome c, caspase 9, caspase 3, MFN2, DRP1, acetylated lysine, p-MAPKAPK-2, MAPKAPK-2 and β-actin were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against SIRT1 and Bax were obtained from Abcam (Abcam, Cambridge, United Kingdom). PGC1α antibody and protein A/G-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, United States). All other chemicals were of analytical grade.
7
Macrophage and Microglia Activation Assay
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RAW264.7 macrophages (RAW, murine), phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich, P1585)-induced THP-1 macrophages (human), and BV-2 microglia cells (murine) were obtained from American Type Culture Collection (Manassas, VA, USA). BMDMs and PMs were harvested as described above. Cells were cultured in RPMI 1640 with 10% heat-inactivated FBS and maintained in a humidified CO2 incubator at 37 °C. To induce pro-inflammatory differentiation, macrophages were treated with 0.5 or 1 μg/mL LPS for up to 24 h. Furthermore, RAW and BMDMs were treated with 0.5 μg/mL LPS (Sigma-Aldrich, L2880) for 12 h in the presence of DMSO (Control), Drp1 inhibitor Mdivi-1 (50 μM; Selleck, S7162), mitochondrial ROS scavenger mitoquinone (MitoQ, 250 and 500 nM; Abmole, M9068) or TLR4 inhibitor resatorvid (TAK-242, 100 nM, Selleck, S7455).
8
Mitochondrial Dynamics Regulation
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SP600125, SB203580, and N-acetyl cysteine (NAC) were obtained from Sigma-Aldrich. Torin1 and Mdivi-1 were purchased from Selleck. Antibodies against mitofusin (MFN)-1, mitofusin (MFN)-2, PARP, caspase3, p62, and β-actin were obtained from Proteintech. Antibodies against dynamin-related protein (Drp)-1 were obtained from ABclonal. Antibodies against cleaved caspase-3, cleaved PARP, and LC3 were obtained from Cell Signaling Technology.
9
Investigating Organelle-Specific Autophagy Pathways
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Antibodies against Drp1, p62, LC3, RIP1, RIP3, TSG101, and CD63 were purchased from Abcam (Cambridge, MA, USA). β-actin, tubulin, and COX IV, which were used as references for the total, cytoplasmic, and mitochondrial fractions, were also acquired from Abcam (Cambridge, MA, USA). Antibodies against Phospho-RIP3 (Thr231/Ser232) and Phospho-Drp1 (Ser616) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Aggresome Detection Kit was acquired from Abcam (Cambridge, MA, USA). MitoTracker Deep Red, the ROS 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit, dihydroethidium (DHE) assay kit, and MitoSOX detection kit were purchased from Invitrogen (Carlsbad, CA, USA). Ad-mRFP-GFP-LC3 was supplied by Beyotime (Shanghai, China). Adenoviral vectors for Drp1 short hairpin RNA (shRNA) and Drp1 mutations (Drp1-S616D and Drp1-S616A) were generated by Genechem Technology (Shanghai, China). Related activators and inhibitors, including Mitochondrial division inhibitor 1 (Mdivi-1), N-acetylcysteine (NAC), and Necrostatin-1 (Nec-1), were obtained from Selleck (Shanghai, China). The Protein A/G Magnetic Beads IP Kit was purchased from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS) and penicillin/streptomycin were procured by Invitrogen (Carlsbad, CA, USA). All other chemicals were supplied by Sigma unless otherwise specified.
10
Oral BCP Suspension Protects CIR Mice
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The BCP suspensions and solvent were given orally according to mice’s body weight once daily for 5 days. The CIR mice model was operated after the final administration. The Sham group was injected with the same volume of saline, and mitophagy inhibitor Mdivi-1 (Selleck Chemicals, TX, USA) was injected intraperitoneally at the onset of reperfusion (3 mg/kg).
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