rat cardiomyocyte cell line h9c2  (ATCC)

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: The Journal of International Medical Research

Article Title: Electron transfer flavoprotein subunit beta suppresses hypoxia/reoxygenation-induced mitochondrial dysfunction and apoptosis in cardiomyocytes

doi: 10.1177/03000605261444866

Figure Lengend Snippet: Hypoxia/reoxygenation (H/R) treatment induces myocardial injury and electron transfer flavoprotein subunit beta (ETFB) downregulation in H9c2 cells. (a) Analysis of cell viability in the control (Con) and H/R model (Model) groups. (b–e) Analysis of myocardial injury markers (creatine kinase–myocardial band, cardiac troponin I, cardiac troponin T, and lactate dehydrogenase) in the Con and Model groups, respectively. (f) Protein expression levels of ETFB, Bcl-2-associated X protein, B-cell lymphoma 2, cysteine-aspartic protease-3, and cleaved cysteine-aspartic protease-6 were detected by western blot after H/R treatment. Data are presented as mean ± SD; each experiment was repeated independently three times (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Con.

Article Snippet: H9c2 cardiomyocytes (RRID:CVCL_0286) were purchased from the Wuhan Procell Life Science & Technology Co., Ltd. (China; Cat# CL-0089) and routinely cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; Cat# 12100046) supplemented with 10% fetal bovine serum (FBS; Gibco, USA; Cat# 10099-141) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Control, Expressing, Western Blot

Journal: The Journal of International Medical Research

Article Title: Electron transfer flavoprotein subunit beta suppresses hypoxia/reoxygenation-induced mitochondrial dysfunction and apoptosis in cardiomyocytes

doi: 10.1177/03000605261444866

Figure Lengend Snippet: Hypoxia/reoxygenation (H/R) treatment induces apoptosis, mitochondrial damage, and oxidative stress in H9c2 cells. (a) Apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (with Cyanine 3-labeled dUTP) in the control (Con) and H/R model (Model) groups. Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (b) The apoptosis rate was quantitatively analyzed via flow cytometry with Annexin V-PE/7-AAD staining. (c) Mitochondrial ultrastructure was observed via transmission electron microscopy (TEM) in the Con and Model groups. Red arrows indicate swollen mitochondria. Green arrows point to blurred cristae. Scale bar, 500 nm. (d–f) The levels of oxidative stress markers, including superoxide dismutase (d), malondialdehyde (e), and glutathione disulfide (f), were measured in the Con and Model groups. All quantitative data (panels a–b, d–f) are presented as mean ± SD from three independent experiments (n = 3). ** p < 0.01, *** p < 0.001 vs. Con.

Article Snippet: H9c2 cardiomyocytes (RRID:CVCL_0286) were purchased from the Wuhan Procell Life Science & Technology Co., Ltd. (China; Cat# CL-0089) and routinely cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; Cat# 12100046) supplemented with 10% fetal bovine serum (FBS; Gibco, USA; Cat# 10099-141) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: TUNEL Assay, Labeling, Control, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy

Journal: The Journal of International Medical Research

Article Title: Electron transfer flavoprotein subunit beta suppresses hypoxia/reoxygenation-induced mitochondrial dysfunction and apoptosis in cardiomyocytes

doi: 10.1177/03000605261444866

Figure Lengend Snippet: Electron transfer flavoprotein subunit beta (ETFB) overexpression suppresses apoptosis and ameliorates mitochondrial morphology in hypoxia/reoxygenation (H/R)-induced H9c2 cells. (a–b) Apoptosis was detected via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (with Cyanine 3-labeled dUTP) in the control (Con), H/R model (Model), empty vector control (Model+NC), and ETFB-overexpressing (Model+ETFB-OE) groups. Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (c–d) The apoptosis rate was quantitatively analyzed via flow cytometry with Annexin V-PE/7-AAD staining in the Con, Model, Model+NC, and Model+ETFB-OE groups. (e) Mitochondrial ultrastructure was observed via transmission electron microscopy (TEM) in the Con, Model, Model+NC, and Model+ETFB-OE groups. Red arrows indicate swollen mitochondria. Green arrows indicate blurred cristae. Scale bar, 500 nm. All quantitative data (panels b and d) are presented as mean ± SD from three independent experiments (n = 3). *** p < 0.001 vs. Con. ns represents not significant vs. Model. ## p < 0.01, ### p < 0.001 vs. Model+NC.

Article Snippet: H9c2 cardiomyocytes (RRID:CVCL_0286) were purchased from the Wuhan Procell Life Science & Technology Co., Ltd. (China; Cat# CL-0089) and routinely cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; Cat# 12100046) supplemented with 10% fetal bovine serum (FBS; Gibco, USA; Cat# 10099-141) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Over Expression, TUNEL Assay, Labeling, Control, Plasmid Preparation, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy

Journal: The Journal of International Medical Research

Article Title: Electron transfer flavoprotein subunit beta suppresses hypoxia/reoxygenation-induced mitochondrial dysfunction and apoptosis in cardiomyocytes

doi: 10.1177/03000605261444866

Figure Lengend Snippet: Electron transfer flavoprotein subunit beta (ETFB) overexpression alleviates oxidative stress in hypoxia/reoxygenation (H/R)-induced H9c2 cells. (a–b) Mitochondrial membrane potential was detected using the JC-1 probe in cells from the following groups: control (Con), H/R model (Model), empty vector control (Model+NC), and ETFB-overexpressing (Model+ETFB-OE). Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (c–d) Intracellular reactive oxygen species levels were assessed using the DCFH-DA probe in the Con, Model, Model+NC, and Model+ETFB-OE groups. Nuclei were counterstained with DAPI. Scale bar, 50 µm (corresponding to a 200× magnification). (e–g) The levels of oxidative stress markers, including superoxide dismutase (e), malondialdehyde (f), and glutathione disulfide (g), were measured in the Con, Model, Model+NC, and Model+ETFB-OE groups. Data are presented as mean ± SD; each experiment was repeated independently three times (n = 3). *** p < 0.001 vs. Con. ns represents not significant vs. Model. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Model+NC.

Article Snippet: H9c2 cardiomyocytes (RRID:CVCL_0286) were purchased from the Wuhan Procell Life Science & Technology Co., Ltd. (China; Cat# CL-0089) and routinely cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, USA; Cat# 12100046) supplemented with 10% fetal bovine serum (FBS; Gibco, USA; Cat# 10099-141) at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Over Expression, Membrane, Control, Plasmid Preparation