nim811  (MedChemExpress)

Journal: The EMBO Journal

Article Title: Mitochondrial calcium regulates lipid metabolism by modulating tethering of mitochondria to lipid droplets

doi: 10.1038/s44318-026-00827-8

Figure Lengend Snippet: ( A ) Super-resolution confocal images pBA in the presence of Valinomycin for the indicated times. Valinomycin induces swelling and PDM detachment. Mitochondria were stained with Live Orange and LDs with BODIPY 493/503 (BD493). ( B ) Representative trace of mt-Ca 2+ influx and efflux in primary adipocytes using mt-GCaMP6. ( C ) Effect of NE on mitochondrial architecture and Δψ m in primary brown adipocytes at different time points. Adipocytes were stained with TMRE (red) and Mitotracker Green (green). Depolarized mitochondria lose red TMRE staining and become green while mitochondria that maintain Δψ m show yellow-orange mitochondrial staining. Note that NE-stimulation results in a mitochondrial shape change from elongated to fragmented and swollen mitochondria. From Wikstrom et al, . ( D , E ) mt-Ca 2+ induced swelling assay in CM and PDM isolated from WT mice using Glutamate+malate as substrates ( n ≥ 6), and in the presence or absence of NIM811, a pharmacological inhibitor of the mitochondrial permeability pore (mt-PTP) modulator, Cyclophylin D (CypD). ( F ) Representative traces of buffer Ca 2+ following the sequential addition of 50 µM bolus of CaCl 2 in respiring isolated CM and PDM. RR is used as a negative control of Ca 2+ entry in mitochondria. ( G ) PC-dependent respiration in BAT CM (blue) and PDM (orange) from NCLX KO mice under the indicated Ca 2+ concentration and in the absence or presence of RR ( n = 3). Note that deletion of NCLX eliminates the differences between PDM and CM shown in Fig. , resulting in CM taking the same Ca 2+ sensitivity phenotype as PDM. ( H ) Testing whether altered sodium concentrations contribute to the observed Ca²⁺ sensitivity due to NCLX activity. State 3 PC-dependent respiration was measured in CM (blue) and PDM (orange) from WT mice under the varying sodium concentrations ( n = 6). Note that changes in sodium concentrations did not impair respiration. Each point represents a biological replica sample. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. .

Article Snippet: NIM811 , Med Chem Express , HY-P0025.

Techniques: Staining, Isolation, Permeability, Negative Control, Concentration Assay, Activity Assay

Journal: The EMBO Journal

Article Title: Mitochondrial calcium regulates lipid metabolism by modulating tethering of mitochondria to lipid droplets

doi: 10.1038/s44318-026-00827-8

Figure Lengend Snippet: ( A , B ) Mitochondrial swelling promotes detachment. ( A ) Mitochondrial swelling assay measured by 540 nm absorbance in isolated WT BAT mitochondria treated with valinomycin in the presence of succinate and rotenone ( n ≥ 3). ( B ) Effect of valinomycin-induced swelling on PDM detachment from the fat layer (FL), assessed using the Reconstituted Detachment Assay (RDA) ( n = 4). Ordinary one-way ANOVA test. ( C , D ) Role of transient permeability transition in mediating detachment. ( C ) RDA performed on BAT FL treated with the Cyclophilin D (CypD) inhibitor NIM811. Mechanically stripped FL (centrifugation) serves as a benchmark of maximal detachment; unstripped FL serves as a negative control ( n = 3). Ordinary one-way ANOVA test. ( D ) In vivo quantification of PDM adherence to LDs in WT and CypD KO BAT from mice housed at room temperature ( n = 4). Unpaired t test. ( E ) Comparison of PDM detachment induced by mechanical stripping versus Ca²⁺-mediated stripping at physiological (10 µM) or toxic (3.4 mM) Ca²⁺ concentrations ( n = 3). Ordinary one-way ANOVA test. ( F – H ) Physiological transient permeability versus irreversible permeability transition. ( F ) Oxygen consumption rates (OCR) of mitochondria stripped mechanically or biochemically. State 3 respiration (under indicated substrates), maximal respiration rate (MRR), and Complex IV-driven OCR were measured ( n = 3). High Ca²⁺ concentrations impair respiratory function. Two-way ANOVA. ( G ) Representative immunoblot and quantification of OPA1 long-to-short (L/S) isoform ratio in mitochondria exposed to 10 µM or 3.4 mM Ca²⁺ ( n = 3). Ordinary one-way ANOVA test. ( H ) Palmitoyl-carnitine (PC)-dependent state 3 respiration in PDM following transient (10 min) versus sustained (40 min) Ca²⁺ exposure, expressed as percentage of untreated control ( n = 3). Ordinary one-way ANOVA test. ( I – K ) Differential Ca²⁺ sensitivity of PDM and CM. ( I ) Ca²⁺-induced swelling kinetics in CM and PDM isolated from WT mice in the presence of PC ( n ≥ 6). Row statistics. ( J ) Quantification of swelling ± NIM811, demonstrating CypD dependence ( n ≥ 5), two-way ANOVA. ( K ) Ca²⁺ retention capacity (CRC) in CM and PDM, shown as representative CaGreen traces following sequential 50 µM CaCl₂ additions. ( L – O ) PDM exhibit heightened sensitivity to Ca²⁺-induced respiratory impairment. ( L ) State 3 PC-driven respiration in CM (blue) and PDM (orange) under increasing Ca²⁺ concentrations ± Ruthenium Red (RR) ( n ≥ 5). ( M ) State 3 PC-driven respiration ± NIM811 ( n = 4). ( N ) OCR in CM and PDM at 1 mM Ca²⁺, normalized to respiration prior to Ca²⁺ addition ( n ≥ 4). RR inhibits Ca²⁺ entry into the matrix. Two-way ANOVA. ( O ) PC-driven respiration in CM and PDM from WT and NCLX KO mice under increasing Ca²⁺ concentrations ( n = 3). Deletion of NCLX abolishes the differential Ca²⁺ sensitivity between CM and PDM observed in WT. ( P , Q ) CypD abundance contributes to differential Ca²⁺ sensitivity. ( P ) Western blot and quantification of CypD distribution in CM and PDM ( n ≥ 3). Two-way ANOVA ( Q ) PC-driven respiration in CM and PDM from CypD KO and WT littermates under increasing Ca²⁺ concentrations ( n = 3). Deletion of CypD eliminates differential Ca²⁺ sensitivity. Data in panels L–M are normalized to untreated controls (no added Ca²⁺). Each point represents a biological replicate; technical replicates were averaged. Reported Ca²⁺ concentrations reflect effective free Ca²⁺ after correction for EGTA chelation in MAS buffer. ( B – J , N – P ) Each point represents a biological replicate; technical replicates were averaged. Data are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. .

Article Snippet: NIM811 , Med Chem Express , HY-P0025.

Techniques: Isolation, Permeability, Centrifugation, Negative Control, In Vivo, Comparison, Stripping Membranes, Western Blot, Control