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: MedComm

Article Title: Loss of Rab8a‐Dependent Tethering of Lipid Droplets to Mitochondria Contributes to Hypoxia–Reoxygenation Injury

doi: 10.1002/mco2.70845

Figure Lengend Snippet: H/R disrupted LD and mitochondria tethering. (A) Representative image of Mitotrakcer (MT) and BODIPY (LD) staining in H9c2 cells subjected to normoxia or H/R. (B) Pearson correlation of MT and LD. (C) Representative image of long chain free fatty acid (LCFA) and BODIPY (LD) staining in H9c2 cells subjected to normoxia or H/R. (D) Pearson correlation of LCFA and LD. (E) Representative image of long chain free fatty acid (LCFA) and Mitotrakcer (MT) staining in H9c2 cells subjected to normoxia or H/R. (F) Pearson correlation of LCFA and MT. (G) Representative image of IMARIS analyzed long chain free fatty acid (LCFA) and BODIPY (LD) staining in H9c2 cells subjected to normoxia or H/R. (H) Quantification of (G). (I) Representative image of IMARIS analyzed long chain free fatty acid (LCFA) and Mitotrakcer (MT) staining in H9c2 cells subjected to normoxia or H/R. (J) Quantification of (I). (K) Seahorse mitochondrial stress test and quantification for parameters of mitochondrial function in H9c2 cells subjected to normoxia or H/R. (L) Quantification of maximal respiration capacity in Seahorse experiment. (M) Representative image of Mitotrakcer (MT) staining of H9c2 cells subjected to normoxia or H/R. (N–P) Quantification of mitochondria density, branch length, and networking. (Q) Representative image of IMARIS analyzed Mitotrakcer (MT) staining of H9c2 cells subjected to normoxia or H/R. (R) Quantification of sphericity of mitochondria. (S) Representative image of long chain free fatty acid (LCFA) and Mitotrakcer (MT) staining in H9c2 cells treated with or without 10 µM Mdivi‐1 and subjected to normoxia or H/R. (T) Pearson correlation of MT and LCFA. Scale bar: 10 µm. Results are presented as mean ± SEM ( n = 3 per group). * p < 0.05, ** p < 0.01, *** p < 0.001 versus normoxia control. One‐way ANOVA was run for statistical analysis. Two‐way ANOVA was run when comparing groups on two different categorical variables.

Article Snippet: The following chemicals, dyes, and antibodies were used: 100 nM rapamycin (Sigma; R8781), 50 μM CQ (Sigma; C6628), 50 μM Baf (Sigma; 196000), 100 μM Para (Sigma; 36186), 10 μM Lalistat 1 (Med Chem Express; HY‐116815), 60 μM etomoxir (Sigma; 236020),1 μM MDIVI‐1 (Med Chem Express; HY‐15886), 20 μM Compound C (Sigma; 171260), 100 nM ALY688 (Allysta Pharmaceutical), 1 mM BODIPY 558/568 C12 (Thermo; D3835), 10 mM BODIPY 493/503 (Thermo; D3922), 100 nM MitoTracker Red (Thermo; M22425 ), 100 nM MitoTracker Green (Thermo; M7514), 300 nM Hoechst (Thermo; H10325 ), GAPDH (Thermo Fisher; MA5‐15738), β‐tubulin (Cell Signaling; 2128), Rab8a (BD Science; 610844), PLIN2 (Proteintech; 60340), PLIN5 (Proteintech; 26951), LC3 (Cell Signaling; 2775), p62 (Cell Signaling; 5114), HIF1⍺ (Cell Signaling; 79233), CPT1⍺ (Proteintech; 15184‐1‐AP), Tom20 (Proteintech; 66777), VDAC (Cell Signaling; 4661), P‐DRP1 Ser616 (Cell Signaling; 3455), P‐DRP1 Ser637 (Cell Signaling; 4867), Parkin (Proteintech; 66674), MFF (Cell Signaling; 84580), Mitofusin 2 (Cell Signaling; 9482), anti‐rabbit (Cell Signaling; 7074), and anti‐mouse (Cell Signaling; 7076).

Techniques: Staining, Control

Journal: Experimental Physiology

Article Title: Therapeutic intervention with sinomenine and irisin to preserve cardiac function after ischaemia–reperfusion injury

doi: 10.1113/EP093586

Figure Lengend Snippet: Proteins related to pyroptosis and mitophagy across experimental groups. Representative western blot bands (a) and protein expression levels of nucleotide‐binding oligomerization domain‐like receptor protein 3 (NLRP3) (b), apoptosis‐associated speck‐like protein containing a CARD (ASC) (c) and gasdermin D N‐terminal (GSDMD‐N) (d) as pyroptosis‐related proteins; and PTEN‐induced putative kinase 1 (PINK1) (e) and Parkin (f) as mitophagy‐related proteins. Data are expressed as means ± standard deviation ( n = 3 per group) and statistically analysed using one‐way ANOVA with Tukey's post hoc test. IR, ischaemia–reperfusion; Iri, irisin; Mdivi‐1, mitochondrial division inhibitor 1; Sin, sinomenine.

Article Snippet: IR + Mdivi‐1 group: Rats were pre‐treated with Mdivi‐1 (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 1 mg/kg/day via intraperitoneal injection for seven consecutive days prior to surgery to pharmacologically inhibit mitophagy, as previously described (Xu & Yu, ).

Techniques: Western Blot, Expressing, Binding Assay, Standard Deviation

Journal: Experimental Physiology

Article Title: Therapeutic intervention with sinomenine and irisin to preserve cardiac function after ischaemia–reperfusion injury

doi: 10.1113/EP093586

Figure Lengend Snippet: Levels of pro‐inflammatory cytokines across experimental groups. Interleukin‐18 (IL‐18) (a) and interleukin‐1β (IL‐1β) (b). Data are expressed as means ± standard deviation ( n = 6 per group) and statistically analysed using one‐way ANOVA with Tukey's post hoc test. IR, ischaemia–reperfusion; Iri, irisin; Mdivi‐1, mitochondrial division inhibitor 1; Sin, sinomenine.

Article Snippet: IR + Mdivi‐1 group: Rats were pre‐treated with Mdivi‐1 (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 1 mg/kg/day via intraperitoneal injection for seven consecutive days prior to surgery to pharmacologically inhibit mitophagy, as previously described (Xu & Yu, ).

Techniques: Standard Deviation

Journal: Experimental Physiology

Article Title: Therapeutic intervention with sinomenine and irisin to preserve cardiac function after ischaemia–reperfusion injury

doi: 10.1113/EP093586

Figure Lengend Snippet: Oxidative stress status across experimental groups. The levels of malondialdehyde (MDA) (a) and the activities of superoxide dismutase (SOD) (b) and glutathione peroxidase (GPx) (c). Data are expressed as means ± standard deviation ( n = 6 per group) and statistically analysed using one‐way ANOVA with Tukey's post hoc test. IR, ischaemia–reperfusion; Iri, irisin; Mdivi‐1, mitochondrial division inhibitor 1; Sin, sinomenine.

Article Snippet: IR + Mdivi‐1 group: Rats were pre‐treated with Mdivi‐1 (MedChemExpress, Monmouth Junction, NJ, USA) at a dose of 1 mg/kg/day via intraperitoneal injection for seven consecutive days prior to surgery to pharmacologically inhibit mitophagy, as previously described (Xu & Yu, ).

Techniques: Standard Deviation