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

Journal: Experimental and Therapeutic Medicine

Article Title: Sotagliflozin ameliorates doxorubicin-induced cardiomyopathy by regulating inflammation and mitochondrial dysfunction through the AMPK/mTOR pathway

doi: 10.3892/etm.2026.13201

Figure Lengend Snippet: SOTA alleviates DOX-induced cardiotoxicity and cardiomyocyte apoptosis in vitro . (A) Cell viability of H9c2 cells treated with DOX (0, 5, 10, 20 and 30 µM) at 12 h. (B) Cell viability of H9c2 cells treated with DOX (10 µM) combined with SOTA (0, 5, 10, 20 and 40 µM) at 12 h. (C) LDH levels in H9c2 cells were detected by a commercial ELISA kit. (D) Representative TUNEL staining images and (E) the quantitative results of H9c2 cells. Scale bar, 20 µm. (F) Statistical results of C cas-3, Bax and Bcl-2 proteins of H9c2 cells and (G) representative images of western blotting. All data are expressed as the mean ± SD (n=3). * P<0.05, ** P<0.01 compared with Control untreated groups, # P<0.05 and ## P<0.01 compared with DOX alone group. ns, not significant; SOTA, sotagliflozin; DOX, doxorubicin; C cas-3, cleaved capsase-3; LDH, lactate dehydrogenase.

Article Snippet: H9c2 cells were obtained from Procell Life Science & Technology Co., Ltd.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Western Blot, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Sotagliflozin ameliorates doxorubicin-induced cardiomyopathy by regulating inflammation and mitochondrial dysfunction through the AMPK/mTOR pathway

doi: 10.3892/etm.2026.13201

Figure Lengend Snippet: SOTA attenuates DOX-induced cardiac inflammation and ROS in vitro . (A) IL-6, IL-1β and TNF-α levels in H9c2 cells as determined by commercial ELISA kits. (B) Representative images of DCFH-DA staining and (C) the quantitative results of ROS levels in H9c2 cells. All data are expressed as the mean ± SD (n=3). Scale bar, 25 µm. ** P<0.01 compared with Control untreated group; ## P<0.01 compared with DOX alone group. SOTA, sotagliflozin; DOX, doxorubicin; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; IL-6, interleukin-6; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: H9c2 cells were obtained from Procell Life Science & Technology Co., Ltd.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Sotagliflozin ameliorates doxorubicin-induced cardiomyopathy by regulating inflammation and mitochondrial dysfunction through the AMPK/mTOR pathway

doi: 10.3892/etm.2026.13201

Figure Lengend Snippet: SOTA attenuates DOX-induced cardiac mitochondrial dysfunction in vitro. (A) Representative images of Rhod2-AM staining and (B) mitochondrial Ca 2+ quantitative results in H9c2 cells. Scale bar, 25 µm . (C) H 2 O 2 levels in H9c2 cells as determined by commercial ELISA kits. (D) Representative images of JC-1 staining and (E) statistical results of JC-1 aggregates to monomers in H9c2 cells. Scale bar, 25 µm . (F) ATP levels in H9c2 cells as determined by ELISA kits. All data are expressed as the mean ± SD (n=3). ** P<0.01 compared with Control untreated group, # P<0.05 and ## P<0.01 compared with DOX alone group. SOTA, sotagliflozin; DOX, doxorubicin; H2O2, hydrogen peroxide; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; DAPI, 4',6-diamidino-2-phenylindole; ATP, adenosine triphosphate.

Article Snippet: H9c2 cells were obtained from Procell Life Science & Technology Co., Ltd.

Techniques: In Vitro, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Sotagliflozin ameliorates doxorubicin-induced cardiomyopathy by regulating inflammation and mitochondrial dysfunction through the AMPK/mTOR pathway

doi: 10.3892/etm.2026.13201

Figure Lengend Snippet: SOTA attenuates DOX-induced cardiomyocyte apoptosis by activating the AMPK/mTOR pathway in vitro . H9c2 cells were treated concomitantly with DOX and SOTA and with 10 µM of the AMPK/mTOR pathway inhibitor compound C. (A) Representative images of western blotting and (B) statistical results of p-AMPK/AMPK and p-mTOR/mTOR proteins in H9c2 cells treated with different concentrations of DOX. (C) Representative images of western blotting and (D) statistical results of p-AMPK/AMPK and p-mTOR/mTOR proteins in the presence of compound C. (E) Statistical results of C cas-3 and (F) Bcl-2/Bax proteins. (G) Quantitative results of H9c2 cells and (H) representative images of TUNEL staining. Scale bar, 20 µm . All data are expressed as the mean ± SD (n=3). * P<0.05, ** P<0.01 compared with DOX group, # P<0.05 and ## P<0.01 compared with SOTA + DOX group. SOTA, sotagliflozin; DOX, doxorubicin; p-, phosphorylated; AMPK, 5' AMP-activated protein kinase; C cas-3, cleaved caspase 3; mTOR, mechanistic target of rapamycin; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: H9c2 cells were obtained from Procell Life Science & Technology Co., Ltd.

Techniques: In Vitro, Western Blot, TUNEL Assay, Staining

Journal: Experimental and Therapeutic Medicine

Article Title: Sotagliflozin ameliorates doxorubicin-induced cardiomyopathy by regulating inflammation and mitochondrial dysfunction through the AMPK/mTOR pathway

doi: 10.3892/etm.2026.13201

Figure Lengend Snippet: SOTA attenuates DOX-induced cardiac mitochondrial dysfunction by activating the AMPK/mTOR pathway in vitro . (A) Representative images of DCFH-DA staining and (B) representative images of JC-1 staining in H9c2 cells. (C) Quantitative results of ROS levels. (D) Quantitative mitochondrial Ca 2+ levels in H9c2 cells. (E) Statistical results of the ratio of JC-1 aggregates to monomers. All data are expressed as the mean ± SD (n=3). ** P<0.01 compared with DOX group, # P<0.05 and ## P<0.01 compared with SOTA + DOX group. SOTA, sotagliflozin; mTOR, mechanistic target of rapamycin; DOX, doxorubicin; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide; DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: H9c2 cells were obtained from Procell Life Science & Technology Co., Ltd.

Techniques: In Vitro, Staining

Journal: PeerJ

Article Title: Post-infarction KLHL40-mediated regulation of cardiac sarcomeric integrity and function

doi: 10.7717/peerj.21375

Figure Lengend Snippet: (A–C) GEO dataset analysis showed that the mRNA expression levels of KLHL40 at sham, 10 min, 1 h, 6 h, 1 day, 3 days, and 7 days after MI. (D) PXD020193 dataset analysis showed that the protein expression levels of KLHL40 at sham, 10 min, 1 h, 6 h, 1 day, and 3 days after MI. (E and F) qRT-PCR and Western blotting semiquantitative analysis of KLHL40 expression in the early stage of human MI. (G and H) qRT-PCR and Western blotting semiquantitative analysis of KLHL40 expression in the late stage of human MI. (I) IHC staining of KLHL40 expression and Masson’s trichrome staining. (J and K) qRT-PCR and Western blotting semiquantitative analyses for KLHL40 in H9C2 cells after 1 h, 3 h, 6 h, 12 h, 18 h, and 24 h hypoxia. ns indicates no significant difference. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The H9C2 cell line was purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in DMEM (#10313021, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining

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