mitotempo  (MedChemExpress)

Journal: The Journal of Experimental Medicine

Article Title: Macrophage anti-bacterial activity is controlled by adenylate kinase 4–mediated mitochondrial DNA synthesis

doi: 10.1084/jem.20250978

Figure Lengend Snippet: Ak4 enhances macrophage antibacterial activities by regulating mtDNA synthesis to boost mtROS production. WT and Ak4 KO TG-pMacs were pretreated with DMSO, dNs, Gem, or ddC at the indicated concentration for 1 h, followed by infection with Listeria at an MOI of 5 for 1 h. Cells were then treated with 250 μg/ml gentamicin, washed with PBS, and maintained in 50 μg/ml gentamicin for 6 h prior to analysis. (A and B) MFI of MitoSox and H 2 DCFDA in WT and Ak4 KO pMacs from Listeria -infected mice for 3 days was analyzed by flow cytometry ( n = 6–10). (C–E) WT and Ak4 KO TG-pMacs were pretreated with DMSO, 1 μM MitoPQ (a mitochondria-targeted redox cycling compound [C]), 200 μM MitoTempo (a mitochondria-targeted antioxidant [D]), or 20 mM NAC (a general cellular antioxidant [E]) for 1 h prior to Listeria infection. Intracellular bacterial loads were assessed by plating cell lysates onto TSA plates and counting CFUs at 24 h after plating ( n = 3–4). (F) MFI of MitoSox in dNs-, Gem-, and ddC-treated WT TG-pMacs after Listeria infection was analyzed by flow cytometry ( n = 4). (G–I) MFI of MitoSox in dNs- (G), Gem- (H), or ddC- (I) treated WT and Ak4 KO TG-pMacs after Listeria infection was analyzed by flow cytometry ( n = 4). (J and K) MFI of MitoSox in dNs- (J) or Gem- (K) treated WT and Ak4 KO TG-pMacs after Salmonella infection was analyzed by flow cytometry ( n = 4). (L) Mock, Ak4 WT, or kinase-dead Ak4 mutants were transduced into Ak4 KO TG-pMacs using lentiviral vectors. Mock-transduced WT TG-pMacs served as controls. MFI of MitoSox from transduced cells after Listeria infection was measured by flow cytometry ( n = 4). Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (A and B) or one-way ANOVA (C–L). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of two independent experiments, and each point represents data from one mouse with two technical repeats. MFI, mean fluorescence intensity.

Article Snippet: The following reagents were used in the study: MitoPQ (a mitochondria-targeted redox cycler, TargetMol), MitoTempo (a mitochondria-targeted antioxidant, TargetMol), NAC(an antioxidant, TargetMol), Gem (a ribonucleotide reductase inhibitor, MedChemExpress), 2′,3′-ddC (a DNA polymerase subunit γ inhibitor, MedChemExpress), and dN mix (MilliporeSigma).

Techniques: Concentration Assay, Infection, Flow Cytometry, Two Tailed Test, Fluorescence

Journal: Scientific Reports

Article Title: Biliary elimination of cholesterol can be modulated by hepatocyte mitochondrial Aquaporin-8 in mice

doi: 10.1038/s41598-026-39058-6

Figure Lengend Snippet: Effect of the mitochondria-targeted antioxidant MitoTempo. AdhAQP8-transduced mice were treated or not with MitoTempo, and the expression of hAQP8, SREBP-2 and ABCG5, and biliary cholesterol excretion were evaluated. (A) Representative immunoblot for hAQP8 in liver mitochondrial fraction, with corresponding densitometric analysis ( n = 4). A 28 kDa immunoreactive band corresponding to human AQP8 is observed. Each lane was loaded with 25 µg of protein. Prohibitin, an inner mitochondrial membrane marker, is shown as the control for equal protein loading. (B) Representative immunoblot for SREBP-2 in total lysates (90 µg protein/lane). Anti-β-actin antibody was used as a control for equal protein loading. Membrane was cut prior to antibody hybridisation based on molecular weight ranges. Densitometric analysis of four separate experiments in each group. (C) Representative immunoblot for ABCG5 in total lysates (90 µg protein/lane). Anti-β-actin antibody was used as a control for equal protein loading. Membrane was cut prior to antibody hybridisation based on molecular weight ranges. Lanes were cropped from non-adjacent regions of the same gel; grouping is indicated by dotted lines. Full-length, uncropped blots are provided in the Supplementary Information. Densitometric analysis of three separate experiments in each group. Data (means ± SEM) are expressed as percentage of controls, n = 3–4. * P < 0.05 from AdhAQP8 without MitoTempo. (D) Effect of MitoTempo on biliary cholesterol excretion in human mtAQP8 mice. Bile was collected in 30-minute period, and cholesterol were determined by thin layer chromatography. Data are given as mean ± standard error ( n = 3–5). Statistical significance was assessed by Student’s t -test comparing AdhAQP8 vs. AdhAQP8 + MitoTempo. * P < 0.05 from AdhAQP8 without MitoTempo.

Article Snippet: MitoTempo was purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Western Blot, Membrane, Marker, Control, Hybridization, Molecular Weight, Thin Layer Chromatography