Journal: Hereditas

Article Title: USP8 protects rat-derived H9C2 cardiomyocytes from doxorubicin-triggered ferroptosis and cell death through deubiquitination-mediated stabilization of MDM4

doi: 10.1186/s41065-025-00527-z

Figure Lengend Snippet: Dox exposure decreases the expression of USP8 and MDM4 in H9C2 cardiomyocytes. ( A ) H9C2 cardiomyocytes were exposed to various concentrations of Dox (0, 0.1, 0.5, 1, 5, and 10 µM) for different time frames (24, 36, and 48 h), and cells were then subjected to viability analysis by CCK-8 assay. ( B and C ) H9C2 cells were stimulated with or without 1 µM Dox for 24 h, and total RNA was prepared for quantitative PCR of USP8 and MDM4 mRNA transcripts ( B ), and cell extracts were collected for immunoblot analysis of USP8 and MDM4 protein levels ( C ). ** P < 0.01, *** P < 0.001

Article Snippet: For MDM4 depletion experiments, an Mdm4 Rat Pre-designed siRNA Set A pool (siMDM4) and a scrambled control (siNC) were used, both sourced from MedChemExpress.

Techniques: Expressing, CCK-8 Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Hereditas

Article Title: USP8 protects rat-derived H9C2 cardiomyocytes from doxorubicin-triggered ferroptosis and cell death through deubiquitination-mediated stabilization of MDM4

doi: 10.1186/s41065-025-00527-z

Figure Lengend Snippet: USP8 mediates MDM4 protein stabilization through its deubiquitinase activity. ( A ) H9C2 cardiomyocytes were exposed to 1 µM Dox or maintained in control medium for 24 h, and cell extracts were harvested for Co-IP assays using anti-MDM4 or anti-IgG antibody. The immunoprecipitates were subjected to immunoblotting (IB) of USP8 and MDM4. ( B ) H9C2 cardiomyocytes transfected with oeNC or oeUSP8 were treated with CHX for 0, 6, and 9 h, and cell extracts were harvested for immunblot analysis of MDM4 and β-actin. ( C ) Untransfected or oeUSP8-transfected H9C2 cardiomyocytes were treated with or without 1 µM Dox for 24 h, and cell extracts were harvested for Co-IP assays using anti-MDM4 antibody. The immunoprecipitates were processed for immunoblotting (IB) of ubiquitin-modified MDM4. ( D ) H9C2 cardiomyocytes with or without transfection were exposed to 1 µM Dox or maintained in control medium for 24 h, and cell extracts were harvested for immunblot analysis of MDM4 protein. ** P < 0.01, *** P < 0.001, ns: non-significant

Article Snippet: For MDM4 depletion experiments, an Mdm4 Rat Pre-designed siRNA Set A pool (siMDM4) and a scrambled control (siNC) were used, both sourced from MedChemExpress.

Techniques: Activity Assay, Control, Co-Immunoprecipitation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Modification

Journal: Hereditas

Article Title: USP8 protects rat-derived H9C2 cardiomyocytes from doxorubicin-triggered ferroptosis and cell death through deubiquitination-mediated stabilization of MDM4

doi: 10.1186/s41065-025-00527-z

Figure Lengend Snippet: MDM4 inhibition reverses the cardioprotective effects of USP8 upregulation against Dox-triggered cell death and ferroptosis in H9C2 cells. ( A ) H9C2 cardiomyocytes were subjected to transfection with siMDM4 or siNC, and cell extracts were collected for immunoblot analysis of MDM4 protein. ( B-I ) H9C2 cardiomyocytes after 24 h transfection with or without oeUSP8 + siNC or oeUSP8 + siMDM4 were stimulated with or without 1 µM Dox for 24 h. ( B ) CCK-8 reagent was added the cells, and absorbance was measured. Viability was determined relative to control cells. ( C ) LDH release was measured in cell supernatants using the assay kit. ( D ) Cells were harvested for flow cytometry after Annexin V/PI staining, and cell death rate per group was quantified. ( E ) Intracellular Fe 2+ levels were determined using a commercial iron assay kit. ( F ) Cells were subjected to BODIPY-C11 probe staining and flow cytometry for the analysis of lipid ROS production. ( G and H ) Cells were processed for MDA and GSH expression quantification using the commercial kits. ( I ) Cell extracts were prepared for immunoblot analysis of GPX4 and SLC7A11. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For MDM4 depletion experiments, an Mdm4 Rat Pre-designed siRNA Set A pool (siMDM4) and a scrambled control (siNC) were used, both sourced from MedChemExpress.

Techniques: Inhibition, Transfection, Western Blot, CCK-8 Assay, Control, Flow Cytometry, Staining, Iron Assay, Expressing