aav9 mmu mir 210 3p inhibition virus  (Genechem)

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: Overexpression of USP18 in the heart exacerbates mitochondrial dysfunction, acute cardiac injury, and cardiac remodeling following I/R in mice. a TTC staining of heart tissue 24 h post-I/R in each group ( n= 5). Scale bar=0.5 cm. ⁎⁎⁎ P <0.001. b Mitochondrial DNA levels ( n= 6) and mitochondrial complexes I and II–III activity ( n= 5) 24 h post-I/R in each group. ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. c Representative electron microscopy images of heart sections 24 h post-I/R. Quantitative analysis of mitochondrial volume density and the percent of mitochondria with cristae loss in each group ( n= 5). Scale bar=10 μm (top) and 6 μm (bottom). Red arrows indicate mitochondria with cristae loss. ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. d Oxygen consumption rate (OCR) and quantitative statistical analysis of basal respiration, ATP-related respiration, maximal respiration, and spare respiratory capacity in mitochondria in the indicated groups ( n= 4). ⁎ P <0.05, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. e H&E staining, PSR staining, and quantitative statistical analysis of cell size and left ventricl e (LV) fibrotic area in AAV9-USP18-transfected mice at 4 weeks after I/R injury ( n= 5). Scale bar=1 mm (left), 50 μm (middle), and 100 μm (right). ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. f Representative B-mode and M-mode echocardiographic images of LV from AAV9-USP18-transfected mice 4 weeks after I/R injury. g Cardiac function of AAV9-USP18-transfected mice after I/R injury at the indicated time points ( n= 6). ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001 vs . AAV9-NC, ns non-significant. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; NC. Negative control; AAV9. Adeno-associated virus serotype 9; AAV9-USP18. Adeno-associated virus serotype 9 encoding USP18; TTC. 2,3,5-triphenyltetrazolium chloride; ATP. Adenosine triphosphate; H&E. Hematoxylin and eosin; PSR. picrosirius red; FCCP. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; LVIDd. Left ventricle internal diameter at diastole; LVISd. Left ventricle internal diameter at systole; LVEF. Left ventricle ejection fraction; LVFS. Left ventricle fractional shortening.

Article Snippet: To induce cardiac-specific USP18 overexpression, male C57BL/6 J mice received a single tail vein injection of adeno-associated virus serotype 9 (AAV9)-cardiac troponin T (cTnT)-USP18 or control AAV9-negative control [60–80 μl, (5.0–6.5)×1013 viral genome/ml, Vigene Bioscience, China] under 1.5%–2% isoflurane anesthesia.

Techniques: Over Expression, Staining, Activity Assay, Electron Microscopy, Transfection, Ubiquitin Proteomics, Negative Control, Virus

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: Parkin knockdown counteracts the protection of USP18 deficiency in vivo. USP18-cKO mice were infected with AAV9-shParkin and subjected to I/R surgery. a Parkin protein levels in mouse hearts infected with AAV9-shParkin ( n= 4). b TTC staining of heart tissue 24 h post-I/R in each group ( n= 5). Scale bar=0.5 cm. c Serum levels of cTnI, CK-MB, and LDH in AAV9-shParkin-infected mice 4 h after I/R surgery ( n= 5). d DNA fragmentation and cleaved caspase-3 activity in heart tissue from AAV9-shParkin-infected mice 24 h after I/R injury ( n= 6). e Oxygen consumption rate (OCR) and quantitative statistical analysis of basal respiration, ATP-related respiration, maximal respiration, and spare respiratory capacity in mitochondria in the indicated groups ( n= 4). f Mitochondrial DNA ( n= 6) and complexes I and II–III activity ( n= 5) 24 h post-I/R in each group. g Representative electron microscopy images of heart sections 24 h post-I/R. The mitochondrial volume density and percent of mitochondria with cristae loss were measured in each group ( n= 5). Scale bar=10 μm (top) and 6 μm (bottom). h Protein levels of P62, ubiquitinated proteins (Ub), and LC3II in mitochondria from heart tissue 24 h after I/R injury ( n= 4). i H&E staining and quantitative statistical analysis of cell size ( n= 5) in USP18-cKO mice and AAV9-shParkin-infected mice 4 weeks after I/R injury. Scale bar=1 μm (top) and 50 μm (bottom). j Heart weight-to-tibia length ratio (HW/TL) ( n= 6) in each group. k Representative B-mode and M-mode echocardiographic images of the left ventricle from USP18-cKO mice and AAV9-shParkin-infected mice 4 weeks after I/R injury. l Cardiac function of USP18-cKO mice and AAV9-shParkin-infected mice 4 weeks after I/R injury ( n= 6). ⁎ P <0.05, ⁎⁎ P <0.01, ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; cTnI. Cardiac Troponin I; CK-MB. Creatine kinase-MB isoenzyme; LDH. Lactate dehydrogenase; NC. Negative control; AAV9. Adeno-associated virus serotype 9; AAV9-USP18. Adeno-associated virus serotype 9 encoding USP18; TTC. 2,3,5-triphenyltetrazolium chloride; ATP. Adenosine triphosphate; H&E. Hematoxylin and eosin; PSR. Picrosirius red; FCCP. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone; LVIDd. Left ventricle internal diameter at diastole; LVISd. Left ventricle internal diameter at systole; LVEF. Left ventricle ejection fraction; LVFS. Left ventricle fractional shortening; HR. Heart rate.

Article Snippet: To induce cardiac-specific USP18 overexpression, male C57BL/6 J mice received a single tail vein injection of adeno-associated virus serotype 9 (AAV9)-cardiac troponin T (cTnT)-USP18 or control AAV9-negative control [60–80 μl, (5.0–6.5)×1013 viral genome/ml, Vigene Bioscience, China] under 1.5%–2% isoflurane anesthesia.

Techniques: Knockdown, In Vivo, Infection, Staining, Activity Assay, Electron Microscopy, Ubiquitin Proteomics, Negative Control, Virus

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 inhibits mitophagy degradation and facilitates cardiac I/R injury through deubiquitinating and upregulating PTEN-L. a The protein levels of PTEN-L and PTEN in USP18-cKO mouse hearts 24 h after I/R injury ( n= 4). ⁎⁎⁎⁎ P <0.0001, ns non-significant. b The protein levels of PTEN-L and PTEN in AAV9-USP18-infected mouse hearts 24 h after I/R injury ( n= 4). ⁎⁎⁎ P <0.001, ⁎⁎⁎⁎ P <0.0001, ns non-significant. c Co-IP of USP18 and PTEN-L in NRVMs (left); NRVMs were transfected with HA-PTEN-L and Flag-USP18 (middle); Co-IP of Flag-USP18 and HA-PTEN-L in NRVMs (right). d Endogenous Co-IP of USP18 and PTEN-L in NRVMs subjected to H/R injury. e Endogenous Co-IP of USP18 and PTEN-L in hearts subjected to I/R injury. f PTEN-L ubiquitination (Ub) levels assessed by CO-IP in NRVMs subjected to H/R injury (top) and hearts subjected to I/R injury (bottom). g NRVMs were transfected with Ad-USP18 or USP18 siRNA or HA-PTEN-L and Myc-Ub and treated with MG132. Co-IP of Myc-Ub and HA-PTEN-L. h Schematic representations of t h e domains of PTEN-L involved in binding to USP18. Full-length PTEN-L or truncated PTEN-L was coexpressed with USP18 in HEK293T cells. Cells were subjected to immunoprecipitation with an anti-Myc antibody or an anti-HA antibody, followed by immunoblotting with the indicated antibodies. i Schematic representations of USP18 residues involved in binding to PTEN-L. Full-length USP18 or USP18 truncations were coexpressed with PTEN-L in HEK293T cells. Cells were subjected to immunoprecipitation with an anti-Myc antibody or an anti-HA antibody, followed by immunoblotting with the indicated antibodies. j Ub assay of PTEN-L in HEK293T cells cotransfected with Myc-USP18, Myc-USP18-mut, and Flag-PTEN-L and treated with 10 μmol/L MG132. k, l NRVMs were transfected with scRNA or USP18 siRNA ( k ), infected with Ad-NC or Ad-USP18 ( l ), and then treated with cycloheximide (CHX, 10 μmol/L) for the indicated time periods. Representative immunoblot analysis of PTEN-L protein levels in each group. ⁎⁎⁎⁎ P <0.0001 vs . scRNA or Ad-NC. USP18. Ubiquitin-specific protease 18; I/R. Ischemia/reperfusion; H/R. Hypoxia-reoxygenation; PTEN. Phosphatase and tensin homolog; PTEN-L. Phosphatase and tensin homolog-long; AAV9. Adeno-associated virus serotype 9; NC. Negative control.

Article Snippet: To induce cardiac-specific USP18 overexpression, male C57BL/6 J mice received a single tail vein injection of adeno-associated virus serotype 9 (AAV9)-cardiac troponin T (cTnT)-USP18 or control AAV9-negative control [60–80 μl, (5.0–6.5)×1013 viral genome/ml, Vigene Bioscience, China] under 1.5%–2% isoflurane anesthesia.

Techniques: Infection, Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics, Binding Assay, Immunoprecipitation, Western Blot, Virus, Negative Control

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Dual Roles of CCN5/WISP2 in Cytosol and Secretome for Maintaining Muscle Homeostasis and Preventing Sarcopenia

doi: 10.1002/jcsm.70282

Figure Lengend Snippet: AAV9‐shCCN5‐mediated CCN5 downregulation produced sarcopenia‐like phenotype in young mice, especially myosteatosis. (A) Scheme of the experimental strategy. (B) Representative images and quantitative data of body composition measurements by dual X‐ray absorptiometry (Green, lean tissue; Red, fat tissue. Scale bars: 1 cm). (C) Representative images and muscle mass of the tibialis anterior (TA) and gastrocnemius (GA). (D) Oil red O staining, immunofluorescent staining of PLIN2 and immunohistochemical staining of adiponectin of GA muscle (scale bars: 50 μm). (E) Total triglyceride concentration in TA muscle. (F) Representative TEM images of lipid droplet (LD) and mitochondria (Mt) in GA muscle and quantification of LD, Mt. (scale bars: 2 [left] and 0.5 [right] μm). The data were presented as mean ± SEM ( n = 5–8/group); * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The adeno‐associated virus 9 (AAV9) employed in this study was designed and produced by Obio Technology Co. Ltd. (Shanghai, China).

Techniques: Produced, Staining, Immunohistochemical staining, Concentration Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Dual Roles of CCN5/WISP2 in Cytosol and Secretome for Maintaining Muscle Homeostasis and Preventing Sarcopenia

doi: 10.1002/jcsm.70282

Figure Lengend Snippet: AAV9‐CCN5‐mediated CCN5 upregulation ameliorated sarcopenia‐like phenotype in aging mice, notably improving myosteatosis. (A) Scheme of the experimental strategy. (B) Representative images and quantitative data of body composition measurements by dual X‐ray absorptiometry (Green, lean tissue; Red, fat tissue. Scale bars: 1 cm). (C) Representative images and muscle mass of the tibialis anterior (TA) and gastrocnemius (GA). (D) HE and WGA staining of GA muscle and quantification of cross‐sectional area (scale bars: 50 μm). (E) Oil red O staining, immunofluorescent staining of PLIN2 and immunohistochemical staining of adiponectin of GA muscle (scale bars: 50 μm). (F) Total triglyceride concentration in TA muscle. The data were presented as mean ± SEM ( n = 6–7/group); * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The adeno‐associated virus 9 (AAV9) employed in this study was designed and produced by Obio Technology Co. Ltd. (Shanghai, China).

Techniques: Staining, Immunohistochemical staining, Concentration Assay