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: The American Journal of Pathology

Article Title: The D2.B10- Dmd mdx /J Mouse Model of Duchenne Muscular Dystrophy Exhibits a Severe Mitochondrial Deficiency Not Observed in the C57BL/10ScSn- Dmd mdx /J Mouse

doi: 10.1016/j.ajpath.2025.09.005

Figure Lengend Snippet: Relative protein expression changes to the latent transforming growth factor (TGF)-β–binding protein 4 (LTBP4) pathway and annexin A6 (ANXA6). LTBP4 is a known genetic modifier in human patients with Duchenne muscular dystrophy and impacts TGF-β signaling. TGF-β acts on SMAD4, which directly interacts with mitochondrial cytochrome c oxidase subunit 2 (MTCO2) and can cause mitochondrial fragmentation via dynamin-related protein 1 (DRP1). A – L: All proteins were assessed for expression levels ( A – F ) relative to total protein ( G – L ). H: TGF-β levels were elevated in D2-WT mice compared with B10-WT mice, and complex IV subunit 2 (MTCO2) was elevated in the D2- mdx mice compared with D2-WT. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Antibodies against latent TGF-β–binding protein 4 (LTBP4; Novus Biologicals, Centennial, CO; NBP2-43671; 1:500), TGF-β (Novus Biologicals; NBP2-46108; 1:2000), annexin A6 (ANXA6; ProteinTech, Rosemont, IL; 68086-1-Ig; 1:2000), mitochondrial cytochrome c oxidase subunit 2 (MTCO2; Thermo Fisher; A-6404; 1:500), SMAD4 (ProteinTech; 10231-1-AP; 1:100), dynamin-related protein 1 (DRP1; ProteinTech; 12957-1-AP; 1:1000), and Total OXPHOS Rodent WB Antibody Cocktail (Abcam; ab110413; 1:25,000) were used.

Techniques: Expressing, Binding Assay

Journal: Nature Communications

Article Title: 1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis

doi: 10.1038/s41467-026-68542-w

Figure Lengend Snippet: HIEC-6 ( a ) were treated with fluorescein labeled polysaccharides and then subjected to immunostaining for Rab5. Scare bar, 10 μm, 2 μm for the enlarged images. The colocalization analysis results are showed in ( b ). HIEC-6 cells were transfected with siRNAs against RAB5A , then treated with polysaccharides. Images of the internalized GFPBW1 and WGE were shown in ( c ), and the quantified results were exhibited in ( e ), and Western blotting results showed the siRNA efficiency ( d ). Data are presented as mean ± SEM from three biologically independent experiments. Unpaired two-tailed t test, * p < 0.05, ** p < 0.01, **** p < 0.0001. Control and RAB5A KO mice ( n = 5 mice for each group) were oral administrated with fluorescein labeled GFPBW1 ( f ) and WGE ( g ), images of the duodenum and jejunum of the mice were shown, the fluorescence intensity was analyzed by Fiji and shown in ( h , i ), respectively. Scare bar, 100 μm, 20 μm for the enlarged images. Data are presented as mean ± SEM from three biologically independent experiments. Unpaired two-tailed t test, *** p < 0.001, **** p < 0.0001. j – l Cells were transfected with RAB5A and the vector plasmid for 48 h, treated with fluorescein labeled GFPBW1 and WGE, and imaged using confocal microscopy, the quantified results are shown in ( l ). k The expression of Rab5 detected by Western blots was shown. Data are presented as mean ± SD from three biologically independent experiments. Unpaired two-tailed t test, * p < 0.05, ** p < 0.01. m , n SPR assay was performed to determine the interaction of polysaccharides and Rab5. Source data are provided as a file.

Article Snippet: A Biacore T200 instrument was used to measure the binding kinetics of human CLTC981-1100aa (abmart, Shanghai, China), human DNM11-271aa (Bioss, Beijing, China), human DNM1 (TP306284, OriGene, USA), human Rab5a (NBC1-18504, Novusbio, USA), human EGFR1-645aa, human Dectin-1, human BMPRIA1-152aa (Sino Biological Inc, Beijing, China) to the polysaccharides.

Techniques: Labeling, Immunostaining, Transfection, Western Blot, Two Tailed Test, Control, Fluorescence, Plasmid Preparation, Confocal Microscopy, Expressing, SPR Assay

Journal: Nature Communications

Article Title: 1,3-and 1,4-linked polysaccharides uptake in intestinal cells relies on clathrin/dynamin 1/Rab5-dependent endocytosis

doi: 10.1038/s41467-026-68542-w

Figure Lengend Snippet: a Colocalization examination was conducted in T24 cells after the incubation of GFPBW1 and WGE for 1 h (green, polysaccharides, red, CLTC), the colocalization analysis results are shown in b . c The interaction of GFPBW1 to living cells in the absence and presence of CLTC antibodies (4 μg/mL). d–f T24 cells were transfected with si- CLTC and scrambled siRNA (Sham). The results of the uptake experiments are presented in d (red, polysaccharides). The efficiency of siRNA was detected by Western blotting ( e ), and the fluorescence intensity was quantified in ( f ). g , h The binding assays of fixed wild-type (Ctrl group) and CLTC-depleted T24 cells (si- CLTC group) to anti-CLTC were performed after the transfection with si- CLTC for 48 h. After regeneration of the chips, an injection of GFPBW1 (100 μg/mL) to both channels performed and the binding curves are displayed in ( h ). The immunostaining results of Rab5 after the treatment of polysaccharides and the colocalization analysis results were presented in i and j , respectively. k Uptake experiments of GFPBW1 and WGE were conducted after the transfection of si- RAB5A , the fluorescence intensity was quantified in l and the efficiency of siRNA was measured by immunoblot ( m ). n–q Further, cells treated with GFPBW1 and WGE were then fixed and stained with dynamin, the immunofluorescence images and the colocalization analysis were shown in ( n ) and o , respectively. p Images of the internalization of polysaccharides in T24 cells after the disturbing of si- DNM1 , the quantified fluorescence result was exhibited in q . Western blotting results showed the siRNA efficiency ( r ). Representative images of the polysaccharide uptake were shown in T24 cells after the treatment of dynasore ( s ), and the fluorescence intensity was quantified in ( t ). Scale bar, 10 μm, 2 μm for the enlarged images. Data are presented as mean ± SEM from three biologically independent experiments. Unpaired two-tailed t -test. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a file.

Article Snippet: A Biacore T200 instrument was used to measure the binding kinetics of human CLTC981-1100aa (abmart, Shanghai, China), human DNM11-271aa (Bioss, Beijing, China), human DNM1 (TP306284, OriGene, USA), human Rab5a (NBC1-18504, Novusbio, USA), human EGFR1-645aa, human Dectin-1, human BMPRIA1-152aa (Sino Biological Inc, Beijing, China) to the polysaccharides.

Techniques: Incubation, Transfection, Western Blot, Fluorescence, Binding Assay, Injection, Immunostaining, Staining, Immunofluorescence, Two Tailed Test