Effects of UBB+1 on oxidative stress-induced cell death.

<p><b><i>A</i></b>, CRT-MG cells stably expressing pEGFP and pEGFP-UBB+1 were incubated in the absence or presence of H₂O₂ (0–100 mmol/L) for different time periods as indicated (0–60 min). After stimulation, cell death was assessed using an LDH assay. Data are presented as the mean ± SEM (<i>n</i> = 3), and Turkey’s post hoc test was applied to significant group effects (<i>p</i><0.0001) identified by ANOVA. Asterisks indicate a significant difference compared with the control: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. <b><i>B</i></b>, Cells were stained with TMRE, and the mean fluorescence intensity was determined using FACS analysis. The graph represents the mitochondrial membrane potential Δψ. Data are presented as the mean ± SEM (<i>n</i> = 3), and Turkey’s post hoc test was applied to significant group effects identified by ANOVA. <b><i>C</i></b>, The mean fluorescence intensity of TMRE staining was assessed after treatment with H₂O₂, and the graph represents the mitochondrial membrane potential Δψ. Data are presented as the mean ± SEM (<i>n</i> = 3), and Turkey’s post hoc test was applied to significant group effects identified by ANOVA. Asterisks indicate a significant difference between UBB+1-expressing and control cells. <b><i>D</i></b>, CRT-MG cells stably expressing pEGFP or pEGFP-UBB+1 were treated with 25 or 50 mmol/L H₂O₂ for 15 min, and mitochondrial membrane potential was assessed using TMRE. Filled, untreated cells; lined, treated cells. <b><i>E</i></b>, CRT-MG cells stably expressing EGFP were transfected with siRNA against Drp1, Mfn1, or the negative control, and stimulated with 0–100 mmol/L H₂O₂ for 0–60 min as indicated. Efficient knockdown of Drp1 and Mfn1 was confirmed by Western blotting. <b><i>F</i></b>, CRT-MG cells stably expressing UBB+1 were transfected with Mfn1 siRNA, control siRNA, Drp1-overexpressing pCMV-Myc or pCMV-Myc. Cells were then stimulated with 0–100 mmol/L H₂O₂ for 30 min. Efficient knockdown of Mfn1 and overexpression of Drp1 were confirmed by Western blotting. After treatment, cell death was assessed by an LDH assay. Data are presented as the mean ± SEM (<i>n</i> = 3); Turkey’s post hoc test was applied to significant group effects (<i>p</i><0.0001) identified by ANOVA. Asterisks indicate a significant difference compared with the control: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. <b><i>G</i></b>, The mitochondrial morphology was analyzed after staining for Tom20 in UBB+1 expressing cells transfected with control siRNA, Mfn1 siRNA, Drp1-overexpressing pCMV-Myc or pCMV-Myc.