Journal: Military Medical Research

Article Title: USP18 exacerbates myocardial I/R injury by inhibiting Parkin mitophagy through the deubiquitinase PTEN-L

doi: 10.1016/j.mmr.2026.100004

Figure Lengend Snippet: USP18 aggravates cardiac I/R injury through regulation of mitochondria and inhibition of mitophagy. a Electron microscopy image showing mitophagy in USP18-cKO mouse hearts ( n= 5). Scale bar=3 μm. White arrowheads indicate sites of mitophagy. b Protein levels of PINK1, Parkin, ubiquitinated proteins (Ub), P62, and LC3II in mitochondria from heart tissue in USP18-cKO and WT mice 24 h after I/R injury ( n =4). c Electron microscopy image showing mitophagy in USP18-overexpres (OV) mouse hearts ( n =5). Scale bar=3 μm. White arrowheads indicate sites of mitophagy. d Protein levels of PINK1, Parkin, Ub, P62, and LC3II proteins in mitochondria from the heart tissue of USP18-OV mice 24 h after I/R injury ( n =4). Color shift in mitophagy dye (red) and lysosomal dye (green) in NRVMs showing mitophagy in NRVMs with USP18 siRNA transfection ( e ) or Ad-USP18 infection ( f ) and the quantitative mitophagy index in each group ( n= 5). Scale bar=9 μm. Protein levels of PINK1, Parkin, Ub, P62, and LC3II in mitochondria from NRVMs transfected with USP18 siRNA ( g ) or infected with Ad-USP18 ( h ). ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001 ⁎⁎⁎⁎ P <0.0001. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; WT. Wild-type; KO. Knockout; P62. Sequestosome 1; LC3. Microtubule-associated protein 1 light chain 3; VDAC. Voltage-dependent anion channel.

Article Snippet: To block mitophagy, the selective dynamin-related protein 1 (Drp1) inhibitor Mdivi-1 was used (50 μmol/L, MedChemExpress, USA).

Techniques: Inhibition, Electron Microscopy, Transfection, Infection, Ubiquitin Proteomics, Knock-Out

Journal: Journal of Virology

Article Title: Bovine coronavirus enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis in a low pH-, dynamin-, cholesterol-, microtubule-, Rab7-, and Rab11-dependent manner

doi: 10.1128/jvi.01274-25

Figure Lengend Snippet: BCoV entry into HRT-18 cells depends on dynamin. ( A ) The maximum safe concentrations of dynasore were determined using the CCK-8 assay. ( B ) Western blot analysis was used to evaluate the BCoV N protein expression levels, with grayscale analysis performed and presented as a bar graph. ( C ) RT-qPCR was performed to assess the BCoV gene copy numbers. ( D ) TCID 50 assay was used to measure the BCoV viral titers in the cell supernatant. ( E ) IFA was used to detect the number of BCoV-infected cells. Scale bar = 100 µm. ( F ) RT-qPCR was used to evaluate the effect of dynasore on the viral entry. ( G ) RT-qPCR was used to evaluate the effect of dynasore on the viral attachment. ( H ) The siRNA silencing efficiency of dynamin was screened; the effects of dynamin-silenced cells on BCoV infection were assessed by ( I ) Western blot, ( J ) RT-qPCR, ( K ) TCID 50 , and ( L ) IFA. ( M ) RT-qPCR was used to evaluate the effect of dynamin-silenced cells on the viral entry; ( N ) RT-qPCR was used to evaluate the effects of dynamin-targeting siRNA on BCoV attachment. Data are presented as the mean ± SD of three independent experiments (not significant, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The inhibitors used in this study included SSAA09E3 (Cat HY-138102, MedChemExpress), a novel inhibitor that blocks the fusion of the viral membrane with the host cell membrane; CPZ (Cat C0982, Sigma), a clathrin-mediated endocytosis inhibitor; nystatin (Cat 475914, Sigma), a caveolae inhibitor that acts as a sterol-binding agent disrupting caveolae; blebbistatin (Cat 203391, Sigma), an inhibitor of micropinocytosis; dynasore (Cat T1848, TargetMol), a dynamin inhibitor; MβCD (Cat T4072, TargetMol), a cholesterol depletion inhibitor; chloroquine (Cat S6999, Selleck) and NH 4 Cl (Cat A9434, Sigma), a potent inhibitor of V-ATPase and a specific inhibitor of acidification of endosomal vesicles; colchicine (Cat HY-16569, MedChemExpress), which inhibits the polymerization of tubulin; E64d (Cat S7393, Selleck), a cathepsin inhibitor; and camostat (Cat HY-13512, MedChemExpress), a TMPRSS2 inhibitor.

Techniques: CCK-8 Assay, Western Blot, Expressing, Quantitative RT-PCR, Infection