Journal: Redox Biology

Article Title: Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H 2 O 2 detoxification

doi: 10.1016/j.redox.2026.104176

Figure Lengend Snippet: Doxorubicin increases mitochondrial H 2 O 2 levels and causes cell death in rat cardiomyocytes. (A) H9c2 cells were treated with the indicated concentrations of DOX for 24 h. Cell death was assessed by Annexin V-FITC staining and analyzed using flow cytometer. Representative histograms of three independent experiments are shown. (B) H9c2 cells were treated with 1 μM DOX for the indicated times. Cell viability was measured using WST-1 reagent. (C-D) H9c2 cells were treated with 1 μM DOX for the indicated times and then stained with 5 μM CM-H 2 DCFDA (C) or 5 μM MitoPY-1 (D) for 15 min. Relative fluorescence intensity (RFI) was measured by flow cytometer. All data are presented as mean ± S.D. (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared to untreated control.

Article Snippet: Rat cardiomyocyte cell line, H9c2 cells (CRL-1446) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM high glucose (SH30022.01, Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic-antimycotic, and 1 μg/ml puromycin.

Techniques: Staining, Flow Cytometry, Fluorescence, Control

Journal: Redox Biology

Article Title: Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H 2 O 2 detoxification

doi: 10.1016/j.redox.2026.104176

Figure Lengend Snippet: PrxⅢ regulates mitochondrial H 2 O 2 accumulation and oxidative damage in DOX-treated cardiomyocytes. (A) H9c2 cells stably expressing either control (pSUPER) or PrxⅢ-targeting siRNA (pSUPER-siPrxⅢ) were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 9 h. Mitochondrial H 2 O 2 levels were assessed using the mitochondria-targeted fluorescent probe MitoPY-1 and visualized by green fluorescence. Scale bar, 75 μm. Five randomly selected microscopic fields per sample were analyzed and combined to represent one biological replicate. (B) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 18 h. FOXO3a localization was assessed by immunofluorescence using anti-FOXO3a (green) antibody and DAPI (blue). Scale bar, 25 μm Five randomly selected microscopic fields per sample were analyzed and combined to represent one biological replicate. All data are expressed as mean ± S.D. Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and ns, p > 0.05.

Article Snippet: Rat cardiomyocyte cell line, H9c2 cells (CRL-1446) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM high glucose (SH30022.01, Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic-antimycotic, and 1 μg/ml puromycin.

Techniques: Stable Transfection, Expressing, Control, Transduction, Fluorescence, Immunofluorescence

Journal: Redox Biology

Article Title: Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H 2 O 2 detoxification

doi: 10.1016/j.redox.2026.104176

Figure Lengend Snippet: PrxⅢ regulates oxidative damage and mitochondrial bioenergetics in DOX-treated cardiomyocytes. (A) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. Cells were labeled with 5 μM 10-NAO and relative fluorescence intensity (RFI) was analyzed by flow cytometer. Representative histograms and quantification are shown. (B) Cells were treated with 1 μM DOX for 12 h and labeled with 10 μM Rho-123 to assess mitochondrial membrane potential ( ΔΨ m ). The percentage of cells with low ΔΨ m was quantified by flow cytometry. Representative histograms and quantification are shown. (C) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. Representative bar graph showing relative ATP concentration normalized to protein contents. (D-H) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 12 h. (D) Representative tracing of the oxygen consumption rate (OCR). Arrows indicate time points when cells were treated with oligomycin, FCCP, and rotenone plus antimycin A (Rot/AA), respectively. The OCR is calculated as (E) basal respiration, (F) maximal respiration, (G) spare respiration, and (H) proton-leak. All data are expressed as mean ± S.D. (n = 3-5). Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Rat cardiomyocyte cell line, H9c2 cells (CRL-1446) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM high glucose (SH30022.01, Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic-antimycotic, and 1 μg/ml puromycin.

Techniques: Transduction, Labeling, Fluorescence, Flow Cytometry, Membrane, Concentration Assay

Journal: Redox Biology

Article Title: Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H 2 O 2 detoxification

doi: 10.1016/j.redox.2026.104176

Figure Lengend Snippet: PrxⅢ regulates mitochondrial biogenesis in DOX-treated cardiomyocytes. (A) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 6 h. Total RNA was isolated and the mRNA levels of PGC-1α, Nrf1, and mtTFA were determined by qRT-PCR. Representative bar graphs showing relative expression normalized to GAPDH are presented. (B) Relative mRNA expression of the mitochondrial DNA-encoded cytochrome c oxidase (COX) mRNA was measured as an indicator of mtDNA copy-dependent transcription. Representative bar graphs showing relative expression normalized to GAPDH are presented. (C) Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 18 h. Representative immunoblots and quantitative analyses of PGC-1α, Nrf1, and mtTFA proteins. GAPDH and α-tubulin were used as loading controls. Densitometric quantification of protein expression was performed from three independent biological replicates. All data are presented as mean ± S.D. (n = 3). Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Article Snippet: Rat cardiomyocyte cell line, H9c2 cells (CRL-1446) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM high glucose (SH30022.01, Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic-antimycotic, and 1 μg/ml puromycin.

Techniques: Transduction, Isolation, Quantitative RT-PCR, Expressing, Western Blot

Journal: Redox Biology

Article Title: Peroxiredoxin Ⅲ safeguards cardiac function against doxorubicin by regulating mitochondrial quality control via H 2 O 2 detoxification

doi: 10.1016/j.redox.2026.104176

Figure Lengend Snippet: PrxⅢ protects cardiomyocytes against DOX-induced apoptotic cell death. Cells were transduced with Ad-PrxⅢ or Ad-Stuffer for 24 h and then exposed to 1 μM DOX for 18 h. (A) Cell lysates were subjected to immunoblot analyses for cleaved Caspase-3 and cleaved PARP-1 with normalization to β-actin. Densitometric quantification of protein expression was performed from three independent biological replicates. (B) Apoptotic cell death was assessed by Annexin V-FITC and 7-AAD double staining followed by flow cytometric analysis. Quantification of Annexin V and/or 7-AAD-positive cells is shown. All data are presented as mean ± S.D. Statistical significance was determined using two-way ANOVA followed by Bonferroni's post hoc test. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: Rat cardiomyocyte cell line, H9c2 cells (CRL-1446) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM high glucose (SH30022.01, Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic-antimycotic, and 1 μg/ml puromycin.

Techniques: Transduction, Western Blot, Expressing, Double Staining