Journal: Scientific Reports

Article Title: Development of hydroxybenzoic-based platforms as a solution to deliver dietary antioxidants to mitochondria

doi: 10.1038/s41598-017-07272-y

Figure Lengend Snippet: Cytotoxicity profile of AntiOxBEN 1 (−) and AntiOxBEN 2 (−) on ( a ) rat embryonic cardiomyoblasts (H9c2), ( b ) human neonatal dermal fibroblasts (HNDF) and ( c ) human hepatocellular carcinoma (HepG2) cells. AntiOxBEN 1 (−) and AntiOxBEN 2 (−) cytotoxicity determined by changes in intracellular ATP levels on ( d ) rat embryonic cardiomyoblasts (H9c2), ( e ) human neonatal dermal fibroblasts (HNDF) and ( f ) human hepatocellular carcinoma (HepG2) cells. Data are means ± SEM of four independent experiments and the results are expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. Statistically significant compared with control group using one-way ANOVA. Significance was accepted with *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Rat embryonic myocardium (H9c2) (ATCC, VA, USA), human hepatocellular carcinoma cells (HepG2) (ECACC, UK) and human neonatal dermal fibroblasts (HNDF) (ATCC, Barcelona, Spain) were used in this study.

Techniques: Control

Journal: Scientific Reports

Article Title: Development of hydroxybenzoic-based platforms as a solution to deliver dietary antioxidants to mitochondria

doi: 10.1038/s41598-017-07272-y

Figure Lengend Snippet: Antioxidant cytoprotective effects of AntiOxBEN 1 and AntiOxBEN 2 on ( a ) rat embryonic cardiomyoblasts (H9c2), ( b ) human neonatal dermal fibroblasts (HNDF) and ( c ) human hepatocellular carcinoma (HepG2) cells against t-BHP-induced metabolic activity decrease. Each compound has three bars, which corresponds to the different concentrations used (from left to right, 25, 50, 100 µM).The comparisons were performed by using one-way ANOVA between the control (t-BHP) vs. preparation where AntiOxBENs were pre-incubated. Data are means ± SEM of four independent experiments and the results are expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. Significance was accepted with *P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Rat embryonic myocardium (H9c2) (ATCC, VA, USA), human hepatocellular carcinoma cells (HepG2) (ECACC, UK) and human neonatal dermal fibroblasts (HNDF) (ATCC, Barcelona, Spain) were used in this study.

Techniques: Activity Assay, Control, Incubation

Journal: Biomolecules

Article Title: Application of Zebrafish Model in the Suppression of Drug-Induced Cardiac Hypertrophy by Traditional Indian Medicine Yogendra Ras

doi: 10.3390/biom10040600

Figure Lengend Snippet: Dose screening and oxidative stress analysis in H9C2 cells exposed to isoproterenol (ISP) and Yogendra Ras (YDR). ( A ) MTT-based dose screening in the H9C2 cells showed a dose-dependent loss of cell viability following exposure to the ISP (IC 20 = 50 µM and IC 50 = 122.4 µM) and YDR (IC 20 = 30 µg/mL and IC 50 = 205 µg/mL). ( B ) Stimulation of the H9C2 cells with ISP (50 µM) induced up-regulation in the production of intracellular reactive oxygen species and its reduction following treatment with YDR. ( C ) Mitochondrial membrane potential (MMP) increment was detected in the ISP-stimulated H9C2 cells. It was reduced following co-treatment with YDR. ( D ) YDR also reduced the ISP-stimulated generation of superoxide ions (O −2 ) in H9C2 cardiomyocytes as indicated through the intracellular formation of formazan in the NBT assay. ( E ) Reduction in the generation of O −2 in the ISP-stimulated H9C2 cells following treatment with YDR was confirmed through the recovery in the intracellular levels of superoxide dismutase (SOD) enzyme. ( F ) Intracellular catalase enzyme levels were also recovered in the ISP-stimulated H9C2 cells following treatment with YDR. All the experiments were performed in biological and technical triplicates. Results represent mean ± SD. The statistical analysis results were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test: p -value # < 0.01 (ISP alone versus normal control) and ** < 0.01, *** < 0.001 (YDR co-treatment versus ISP alone).

Article Snippet: The H9C2 (rat embryonic cardiac) cells were purchased from the ATCC licensed cell repository National Centre for Cell Science (NCCS), Pune, India.

Techniques: Membrane, Control

Journal: Biomolecules

Article Title: Application of Zebrafish Model in the Suppression of Drug-Induced Cardiac Hypertrophy by Traditional Indian Medicine Yogendra Ras

doi: 10.3390/biom10040600

Figure Lengend Snippet: Down-regulation of cardiac-hypertrophy-associated non-clinical and clinical molecular biomarkers by Yogendra Ras (YDR) in the isoproterenol (ISP)-stimulated H9C2 cells. H9C2 cells stimulated with ISP showed an increase in the mRNA expression levels of the pro-inflammatory mediator genes: ( A ) Cyclooxygenase-2 (COX-2), ( B ) NADPH oxidase-2 (NOX-2). and ( C ) NADPH oxidase-4 (NOX-4) using quantitative real-time PCR. Significant down-regulation of these pro-inflammatory genes was observed following a co-treatment of the ISP-stimulated H9C2 cells with YDR. Treatment of the H9C2 cells with ISP also induced an up-regulation of the expression of the fetal cardiac fetal gene ( D ) atrial natriuretic factor (ANF) and ( E ) cardiolipin (CRLS-1), ( F ) troponin I (TNN-I), and ( G ) troponin T (TNN-T). Expression of all the genes was normalized against the house-keeping gene β-actin. Co-treatment of the ISP-stimulated H9C2 cells with YDR significantly reduced the expression of all the up-regulated non-clinical and clinical genes. H) The heat map was generated to summarize the gene expression study. All the experiments were performed in biological and technical triplicates. Results represent mean ± SD. For the statistical analysis, results were analyzed using one-way ANOVA followed by Dunnett’s post-hoc test: p -value # < 0.01 (ISP/YDR versus normal control) and * < 0.05, **< 0.01 (YDR co-treatment versus ISP alone).

Article Snippet: The H9C2 (rat embryonic cardiac) cells were purchased from the ATCC licensed cell repository National Centre for Cell Science (NCCS), Pune, India.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Generated, Gene Expression, Control

Journal: Antioxidants

Article Title: Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue

doi: 10.3390/antiox12091703

Figure Lengend Snippet: Effect of H 2 O 2 on desmin wt or C333S network organization and oligomerization in H9c2 cardiomyoblasts. Cells were transfected with desmin wt or C333S and treated with 1 mM H 2 O 2 for 2 h, as indicated. ( A ) Non-transfected H9c2 cells show undetectable levels of desmin either by immunofluorescence ( upper panel) or by western blot ( lower panel). NT, non-transfected cells; Desmin WT, cells transfected with desmin wt. ( B ) Cells grown on coverslips were fixed and stained for desmin and f-actin, and nuclei were counterstained with DAPI. Overall projections showing the distribution of desmin ( left images) and the overlays with f-actin and DAPI ( right images) are shown. Bars, 25 µm. ( C ) The graph displays the ratios between the area of the cells with detectable desmin filaments and that occupied by f-actin. The results shown are average values from at least 15 determinations ± SEM. * p < 0.05 by Student’s t -test. ( D ) Cells on cell culture wells were lysed, proteins were analyzed by SDS-PAGE under non-reducing or reducing conditions, as indicated, and desmin was detected by western blot. The results are representative of four experiments. Dotted lines indicate where lanes from the same gel have been cropped.

Article Snippet: H9c2 cardiomyoblasts (rat heart myoblasts derived from embryonic BD1X rat heart myocardium) from the European Collection of Authenticated Cell Cultures (ECACC General Cell Collection, originally deposited by the ATCC) were cultured in DMEM plus antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin), supplemented with FBS (10% v / v , Gibco, New York, NY, USA), routinely in a humidified atmosphere with 5% CO 2 .

Techniques: Transfection, Immunofluorescence, Western Blot, Staining, Cell Culture, SDS Page

Journal: Antioxidants

Article Title: Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue

doi: 10.3390/antiox12091703

Figure Lengend Snippet: Effect of 15d-PGJ 2 on desmin network organization and protein levels in H9c2 cardiomyoblasts. Cells transfected as above were treated with 10 µM 15d-PGJ 2 for 2 h. ( A ) Distribution of desmin and f-actin was assessed as in . ( B ) Ratios between the cell areas covered by detectable structures of desmin and actin. Results shown are average values from at least 18 determinations ± SEM. *** p < 0.001, ** p < 0.01 by Student’s t -test. ( C ) Proportion of cells showing desmin accumulations. Results are average values ± SEM from four different assays totaling between 50 and 100 cells per experimental condition. ( D ) Lysates from cells treated as in ( A ) were analyzed by gel electrophoresis under non-reducing conditions, and desmin was detected by western blot. Results are representative of four experiments. The dotted line indicates where lanes from the same gel have been cropped.

Article Snippet: H9c2 cardiomyoblasts (rat heart myoblasts derived from embryonic BD1X rat heart myocardium) from the European Collection of Authenticated Cell Cultures (ECACC General Cell Collection, originally deposited by the ATCC) were cultured in DMEM plus antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin), supplemented with FBS (10% v / v , Gibco, New York, NY, USA), routinely in a humidified atmosphere with 5% CO 2 .

Techniques: Transfection, Nucleic Acid Electrophoresis, Western Blot

Journal: Antioxidants

Article Title: Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue

doi: 10.3390/antiox12091703

Figure Lengend Snippet: Effect of chemical hypoxia on the organization of desmin wt or C333S in H9c2 cells. Cells were transfected with desmin wt or C333S, as indicated, and treated with the specified concentrations of CoCl 2 for 24 h. ( A ) Cells were fixed and processed for detection of desmin (immunofluorescence), f-actin (phalloidin-TRITC staining), and nuclei (DAPI). ( B ) The proportion of cells showing any type of desmin condensation or aggregate was obtained by visual inspection from three different experiments. Results shown are average values ± SEM. * p < 0.05. Bars, 20 µm. ( C ) Lysates from cells treated as in ( A ) were analyzed by SDS-PAGE under non-reducing conditions and immunoblotted with an anti-desmin antibody. Results are representative of three assays with similar observations.

Article Snippet: H9c2 cardiomyoblasts (rat heart myoblasts derived from embryonic BD1X rat heart myocardium) from the European Collection of Authenticated Cell Cultures (ECACC General Cell Collection, originally deposited by the ATCC) were cultured in DMEM plus antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin), supplemented with FBS (10% v / v , Gibco, New York, NY, USA), routinely in a humidified atmosphere with 5% CO 2 .

Techniques: Transfection, Immunofluorescence, Staining, SDS Page

Journal: Antioxidants

Article Title: Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue

doi: 10.3390/antiox12091703

Figure Lengend Snippet: Importance of C333 in desmin assembly in several cellular models. ( A ) H9c2 cardiomyoblasts were transfected with mEmerald-desmin wt or C333S, as indicated. The morphology of the desmin network was assessed 48 h later by direct fluorescence visualization; f-actin was stained with Phalloidin-TRITC, and nuclei were counterstained with DAPI. The proportion of cells displaying desmin aggregates is shown in ( B ). Results are average values ± SEM from three different experiments. ** p < 0.01 by Student’s t -test. ( C ) SW13/cl.2 cells were transfected with the indicated plasmids, and the distribution of the desmin fluorescent construct was assessed by confocal microscopy. Images are representative of four assays with similar results. ( D ) SW13/cl.2 cells expressing desmin wt or C333S were treated with H 2 O 2 , as indicated. The morphology of the desmin network was assessed by immunofluorescence. The proportion of cells showing extended desmin filaments is shown in ( E ). Results are average values ± SEM from three independent experiments. * p < 0.05 by Student’s t -test.

Article Snippet: H9c2 cardiomyoblasts (rat heart myoblasts derived from embryonic BD1X rat heart myocardium) from the European Collection of Authenticated Cell Cultures (ECACC General Cell Collection, originally deposited by the ATCC) were cultured in DMEM plus antibiotics (100 U/mL penicillin, 100 μg/mL streptomycin), supplemented with FBS (10% v / v , Gibco, New York, NY, USA), routinely in a humidified atmosphere with 5% CO 2 .

Techniques: Transfection, Fluorescence, Staining, Construct, Confocal Microscopy, Expressing, Immunofluorescence

Journal: Biomolecules

Article Title: Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury

doi: 10.3390/biom9110678

Figure Lengend Snippet: Schematic diagram of the experimental design. Vascular endothelial cells (EC) (EA.hy926) were stably transfected by a eukaryotic expression vector containing human secretory leukocyte protease inhibitor (SLPI) mRNA ( hspli ) ( A ). The stable cells overexpressing recombinant human SLPI were used for further experiments. Determination of the paracrine effect of endothelial-derived SLPI on cardiomyocyte (CM) (H9c2) subjected to hypoxia/reoxygenation (H/R) injury was performed via techniques following two different principles: co-culture between SLPI-overexpressing EA.hy926 cells and H9c2 cells by the Transwell culture system ( B ), and conditioned medium from SLPI-overexpressing EA.hy926 cells ( C ).

Article Snippet: The human umbilical vein cell line (EA.hy926) and rat embryonic cardiomyocyte cell line (H9c2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) as ATCC-CRL2992 and ATCC-CRL1446, respectively.

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Protease Inhibitor, Recombinant, Derivative Assay, Co-Culture Assay

Journal: Biomolecules

Article Title: Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury

doi: 10.3390/biom9110678

Figure Lengend Snippet: The effect of rhSLPI secreted from the endothelial cell line protected the cardiomyocyte cell line from hypoxia/reoxygenation. ( A ) Wild-type (EA-WT) and SLPI-overexpressing (EA-SLPI) EA.hy926 cells were co-cultured with the H9c2 cell line at ratios 1:1 and 1:3, then subjected to H/R, and the cell viability was determined by MTT; ( B ) The culture media from wild-type (EA-WT) and SLPI-overexpressing (EA-SLPI) EA.hy926 cells were transferred to culture H9c2 cells at ratios of 1:1 and 1:3; then, the cells were exposed to H/R and the cell viability was determined by MTT. After transferring the media from wild-type (EA-WT) and SLPI-overexpressing (EA-SLPI) EA.hy926 cells at ratios of 1:1 and 1:3 to the H9c2 cells, ( C ) the cells were exposed to H2DCFDA followed by H/R, and the intracellular ROS levels were determined by spectrophotometry. Each bar represents the mean ± SEM. * p < 0.05 vs. the H9c2 not subjected to H/R (ANOVA), # p < 0.05 vs. the H9c2 subjected to H/R (ANOVA), † p < 0.05 vs. the 1:1 or 1:3 culture ratio group (ANOVA, n =3).

Article Snippet: The human umbilical vein cell line (EA.hy926) and rat embryonic cardiomyocyte cell line (H9c2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) as ATCC-CRL2992 and ATCC-CRL1446, respectively.

Techniques: Cell Culture, Transferring, Spectrophotometry

Journal: Biomolecules

Article Title: Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury

doi: 10.3390/biom9110678

Figure Lengend Snippet: Determination of the effect of rhSLPI secreted from endothelial cells on the cardiomyocyte cell line subjected to H/R in terms of the cellular signaling response. The media from cultured wild-type (EA-WT) and SLPI-overexpressing (EA-SLPI) EA.hy926 cells were transferred to culture H9c2 cells; then, the cells were exposed to H/R, and the activation of Akt ( A ) and p-38 MAPK ( B ) was determined by Western blot analysis. Each bar represents the phosphorylation ratio (phospho/total) of Akt and p38 MAPK. * p < 0.05 vs. H9c2 not subjected to H/R (ANOVA), # p < 0.05 vs. H9c2 subjected to H/R (ANOVA), † p < 0.05 vs. the H9c2/EA-WT medium group (ANOVA, n = 3).

Article Snippet: The human umbilical vein cell line (EA.hy926) and rat embryonic cardiomyocyte cell line (H9c2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) as ATCC-CRL2992 and ATCC-CRL1446, respectively.

Techniques: Cell Culture, Activation Assay, Western Blot, Phospho-proteomics

Journal: Biomolecules

Article Title: Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury

doi: 10.3390/biom9110678

Figure Lengend Snippet: Determination of the effect of rhSLPI secreted from endothelial cells on the cardiomyocyte cell line subjected to H/R in terms of the apoptosis pathway. The media from cultured wild-type (EA-WT) and SLPI-overexpressing (EA-SLPI) EA.hy926 cells were transferred to culture H9c2 cells; then, the cells were exposed to H/R, and the apoptotic proteins, including Bax/Bcl-2 ( A ), caspase-3 ( B ), and caspase-8 ( C ), were determined by Western blot analysis. Each bar represents the ratio relative to the reference protein (GAPDH). * p < 0.05 vs. H9c2 not subjected to H/R (ANOVA), # p < 0.05 vs. H9c2 subjected to H/R (ANOVA, n = 3).

Article Snippet: The human umbilical vein cell line (EA.hy926) and rat embryonic cardiomyocyte cell line (H9c2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) as ATCC-CRL2992 and ATCC-CRL1446, respectively.

Techniques: Cell Culture, Western Blot

Journal: Biomolecules

Article Title: Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury

doi: 10.3390/biom9110678

Figure Lengend Snippet: A schematic diagram of the major findings in this study. The rhSLPI that was overexpressed and secreted from vascular endothelial cells could protect not only endothelial cells but also cardiomyocytes from I/R injury. The mechanisms for this could be due to the attenuation of intracellular ROS production, activation of Akt and p38 MAPK phosphorylation, and reduction of apoptotic regulatory proteins (Bcl-2, caspase-3, caspase-8).

Article Snippet: The human umbilical vein cell line (EA.hy926) and rat embryonic cardiomyocyte cell line (H9c2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) as ATCC-CRL2992 and ATCC-CRL1446, respectively.

Techniques: Activation Assay, Phospho-proteomics

Journal: Mediators of Inflammation

Article Title: lncRNA DLX6-AS1 Promotes Myocardial Ischemia-Reperfusion Injury through Mediating the miR-204-5p/FBXW7 Axis

doi: 10.1155/2023/9380398

Figure Lengend Snippet: DLX6-AS1 interacted with miR-204-5p and repressed miR-204-5p expression. (a) The predicted binding sites between DLX6-AS1 and miR-204-5p were presented. (b) The interaction between DLX6-AS1 and miR-204-5p in H9c2 cells was verified by luciferase reporter assay. (c) IR rats were established by LAD. Sham-operated rats were served as control. H9c2 cells were subjected to HR. Normal H9c2 cells were served as control. The expression of miR-204-5p and DLX6-AS1 in the myocardial tissues and H9c2 cells was examined by qRT-PCR. (d) RIP assay was carried out utilizing an anti-Ago2 antibody in H9c2 cells transfected with pc-DLX6-AS1 to detect the expression of DLX6-AS1 and miR-204-5p through qRT-PCR. (e) The qRT-PCR was carried out to assess the expression of miR-204-5p in H9c2 cells transfected with si-NC, si-DLX6-AS1, pc-DNA, or pc-DLX6-AS1. ∗∗ P vs. NC-mimic, Sham, Control, IgG, si-NC; ## P vs. pc-DNA.

Article Snippet: The rat embryonic cardiomyocyte cell line H9c2 (CCTCC, Wuhan, China) was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 μ g/mL penicillin/streptomycin at 37°C and 5% CO 2 .

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Control, Quantitative RT-PCR, Transfection

Journal: Mediators of Inflammation

Article Title: lncRNA DLX6-AS1 Promotes Myocardial Ischemia-Reperfusion Injury through Mediating the miR-204-5p/FBXW7 Axis

doi: 10.1155/2023/9380398

Figure Lengend Snippet: DLX6-AS1 promoted FBXW7 expression by sponging miR-204-5p. (a) The predicted binding sites between miR-204-5p and FBXW7 were presented. (b) The interaction between miR-204-5p and FBXW7 in H9c2 cells was verified by luciferase reporter assay. (c) H9c2 cells were transfected with pc-DLX6-AS1 or pc-DNA and miR-204-5p-mimic or NC-mimic. FBXW7 expression in the H9c2 cells was examined by WB. ∗∗ P vs. NC-mimic, pc-DNA + NC-mimic; ## P vs. pc-DNA + miR-204-5p-mimic; && P vs. miR-204-5p-mimic + pc-DLX6-AS1.

Article Snippet: The rat embryonic cardiomyocyte cell line H9c2 (CCTCC, Wuhan, China) was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 μ g/mL penicillin/streptomycin at 37°C and 5% CO 2 .

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Transfection

Journal: Mediators of Inflammation

Article Title: lncRNA DLX6-AS1 Promotes Myocardial Ischemia-Reperfusion Injury through Mediating the miR-204-5p/FBXW7 Axis

doi: 10.1155/2023/9380398

Figure Lengend Snippet: DLX6-AS1 deficiency repressed inflammatory response and cell apoptosis in HR-treated H9c2 cells by regulating miR-204-5p. H9c2 cells were transfected with si-DLX6-AS1 or si-NC and miR-204-5p inhibitor or inh-NC, followed by HR treatment. (a) The expression of MCP-1, IL-6, and IL-1 β in the H9c2 cells was detected by qRT-PCR. (b) The expression of Bax and Bcl-2 in the H9c2 cells was assessed by WB. (c) Apoptosis of H9c2 cells was measured through flow cytometry. ∗∗ P vs. si-NC + inh-NC; ## P vs. si-NC + miR-204-5p inhibitor; && P vs. miR-204-5p inhibitor + si-DLX6-AS1.

Article Snippet: The rat embryonic cardiomyocyte cell line H9c2 (CCTCC, Wuhan, China) was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 μ g/mL penicillin/streptomycin at 37°C and 5% CO 2 .

Techniques: Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) H9C2 cells were fixed and stained for mitochondria (in green) or for PI5P (in green). Zoomed region (i) and (ii) are shown below. (B) Line scan of fluorescence intensity along each arrow from inserts (i) and (ii) from (A). (C) Proportion of mitochondrial vs. non-mitochondrial PI5P. Results are from 19 cells across 3 independent experiments. (D) Cells were treated with STA (100nM for the indicated time) or with DMSO (vehicle only), fixed and stained for mitochondria (in green) or PI5P (in red). Right panel show the quantification of PI5P fluorescence intensity in the mitochondrial (mito.) or non-mitochondrial mask (non-mito.). Results are from 13-27 cells across 3 independent experiments. Statistical significance was determined by unpaired t-test. *p<0.05, ***p < 0.001.

Article Snippet: The rat embryonic cardiomyoblastic cell line H9C2 (2-1, Merck) was cultured in DMEM medium (31966, ThermoFisher Scientific) supplemented with 10% FBS and 1% penicillin– streptomycin in a 37°C, 5% CO2 incubator.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: The phosphoinositide PI5P impairs mitochondrial function through endosome-mitochondria proximity

doi: 10.1101/2025.09.22.677701

Figure Lengend Snippet: (A) Schematic depicting the subcellular fractionation performed on H9C2 cells to obtained a post-nuclear supernatant (PNS), supernatant (SPNT) and pellet fractions. (B) The PNS, SPNT and pellet fractions were immunoblotted with the indicated antibodies. Quantification was performed from 4-8 independent experiments. (C) The pellet fractions obtained from control or 2DG-treated cells were immunoblotted with the indicated antibodies. Quantifications are shown on the right. Statistical significance was determined by unpaired t-test. *p<0.05, **p < 0.01, ns non-significant.

Article Snippet: The rat embryonic cardiomyoblastic cell line H9C2 (2-1, Merck) was cultured in DMEM medium (31966, ThermoFisher Scientific) supplemented with 10% FBS and 1% penicillin– streptomycin in a 37°C, 5% CO2 incubator.

Techniques: Fractionation, Control

Journal: BMC cardiovascular disorders

Article Title: Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury via PI3K/AKT pathway mediated HIF-1α to regulate the mitochondrial dynamic balance.

doi: 10.1186/s12872-024-03868-1

Figure Lengend Snippet: Fig. 1 Effect of H/R injury, SpostC and PI3K/AKT inhibitors on H9C2 cells. (A)Cell viability. (B) Cell survival rate. (C, D) Flow cytometry to measure apoptosis *: Compared with Control group, P < 0.05; **: Compared with Control group, P < 0.01; ***: Compared with Control group, P < 0.001; &: Compared with H/R group, P < 0.05; &&: Compared with H/R group, P < 0.01; &&&: Compared with H/R group, P < 0.001; ^^^: Compared with H/R + SpostC group, P < 0.001

Article Snippet: The H9C2 rat embryonic cardiomyocyte line was obtained from keyGEN BioTECH.

Techniques: Flow Cytometry, Control

Journal: BMC cardiovascular disorders

Article Title: Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury via PI3K/AKT pathway mediated HIF-1α to regulate the mitochondrial dynamic balance.

doi: 10.1186/s12872-024-03868-1

Figure Lengend Snippet: Fig. 3 Fluorescence intensity histogram of mitochondria. (A) Representative images of Mito Tracker™ Red were capture using a laser scanning confocal microscope in H9C2 cells (200×). (B)The Histogram of mitochondrial fluorescence intensity in H9C2 cells. **: Compared with Control group, P < 0.01; ***: Compared with Control group, P < 0.001; &&: Compared with H/R group, P < 0.01; ^: Compared with H/R + SpostC group, P < 0.05

Article Snippet: The H9C2 rat embryonic cardiomyocyte line was obtained from keyGEN BioTECH.

Techniques: Fluorescence, Microscopy, Control

Journal: BMC Complementary Medicine and Therapies

Article Title: Cardioprotective effects of polydatin against myocardial injury in diabetic rats via inhibition of NADPH oxidase and NF-κB activities

doi: 10.1186/s12906-020-03177-y

Figure Lengend Snippet: Polydatin prevents NADPH oxidase activity in HG-induced H9c2 cells. H9c2 cells were cultured in DMEM containing 30 mmol/L glucose (HG) or 5.5 mmol/L glucose (normal glucose) and treated with polydatin (0–40 μmol/L) for 24 h. a Cell viability was measured by a Cell Counting Kit-8 assay. b Cytotoxicity was detected via lactate dehydrogenase (LDH) assay. c NADPH oxidase activity was measured by lucigenin chemiluminescence’s assay. d Intracellular superoxide anion levels were detected in H9c2 cells using fluorogenic probe DCFH-DA. e , f and g Summary of real-time PCR data showing levels of NOX2, NOX4, and NOX1 mRNA in H9c2 cells. Data were expressed as means ± SEM ( n = 6–7). *P < 0.05, **P < 0.01 versus control values; #P < 0.05, ##P < 0.01versus HG

Article Snippet: Embryonic rat cardiac H9c2 cells were provided by Costar Corning Inc. (Corning, CA, USA) and cultured in DMEM Medium with 5.5 mmol/L glucose (normal glucose) added with 10% fetal bovine serum in a 5% CO 2 humidified atmosphere at 37 °C.

Techniques: Activity Assay, Cell Culture, Cell Counting, Lactate Dehydrogenase Assay, Real-time Polymerase Chain Reaction, Control

Journal: BMC Complementary Medicine and Therapies

Article Title: Cardioprotective effects of polydatin against myocardial injury in diabetic rats via inhibition of NADPH oxidase and NF-κB activities

doi: 10.1186/s12906-020-03177-y

Figure Lengend Snippet: Polydatin inhibits NF-κB activation in HG-induced H9c2 cells. H9c2 cells were cultured in DMEM containing 30 mmol/L glucose (HG) or 5.5 mmol/L glucose (normal glucose) and treated with polydatin (40 μmol/L), DPI (a NADPH oxidase inhibitor, 10 μmol/L) or polydatin (40 μmol/L) + DPI (10 μmol/L) for 24 h. The expression of TNF-α, IL-1β, IL-6 and VCAM-1 was determined by ELISA. NF-κB expression was assessed by Western blot. a IL-1β levels in H9c2 cells. b IL-6 levels in H9c2 cells. c TNF-α levels in H9c2 cells. d VCAM-1 levels in H9c2 cells. e The expression of p65 in nucleus of H9c2 cells. f The expression of phosphorylation of IκB-α in cytoplasm of H9c2 cells. g Representative immunoblots of p65 and phosphorylation of IκB-α protein levels in H9c2 cells. Results were expressed as means ± SEM (n = 6). *P < 0.05, **P < 0.01 versus control values; #P < 0.05, ##P < 0.01versus HG

Article Snippet: Embryonic rat cardiac H9c2 cells were provided by Costar Corning Inc. (Corning, CA, USA) and cultured in DMEM Medium with 5.5 mmol/L glucose (normal glucose) added with 10% fetal bovine serum in a 5% CO 2 humidified atmosphere at 37 °C.

Techniques: Activation Assay, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Control

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: H9c2 cell viability after 24-hour exposure to N-acetylcysteine (NAC) was assessed by MTT assay (mean + standard deviation (SD) from 4 independent experiments). * p < 0.05 compared with control.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: MTT Assay, Standard Deviation, Control

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: H9c2 cell viability after (A) 24-hour and (B) 48-hour exposure to CY alone and CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9) was assessed by MTT assay (mean + SD from 3 independent experiments). (C) The changes of CY and its metabolites HCY and CEPM concentration in H9c2 cell culture media exposed to CYS9 (mean + SD from 3 independent experiments). (D) Fluorescence intensities, corresponding to levels of H 2 O 2 , in control samples or cells exposed to 250 μM CY, S9, CYS9 for 1 hour (mean + SD from 3 independent experiments). Fluorescence intensity is shown in arbitrary units. * p < 0.05 compared with control.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: MTT Assay, Concentration Assay, Cell Culture, Fluorescence, Control

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: (A) The effect of candidate cardioprotectant agents (NAC, isorhamnetin (ISO), and β-ionone (BIO)) on cytotoxicity of CYS9 in H9c2 cells after 24-hour exposure. (mean + SD from 2 independent experiments conducted in duplicate). * p < 0.05 compared with CYS9 group. (B) The effects of candidate cardioprotectant agents against LDH release from H9c2 cells exposed to CYS9 for 2 hours. (mean + SD from 3 independent experiments). * p < 0.05 compared with CYS9 group.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques:

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: H9c2 cells were exposed to CYS9 for 1 or 2 hours with and without candidate cardioprotectant agents (NAC, ISO, and BIO). Changes in concentration in H9c2 cell culture media of (A) CY and its metabolites (B) HCY and (C) CEPM was evaluated using LC/MS/MS. (mean + SD from 3 independent experiments). * p < 0.05 compared with CYS9 group.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: Concentration Assay, Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: (A) H9c2 cells were exposed for 1 and 2 hours to CYS9 with and without NAC. The changes of acrolein in culture media was measured using HPLC. (mean + SD from 3 independent experiments). Effect of NAC on ROS generated by CYS9, as shown by fluorescence intensity of (B) DCFH, (C) APF, and (D) HPF in cells exposed for 1 hour to CYS9 or CYS9 plus NAC. Fluorescence intensity is shown in arbitrary units. (mean + SD from 3 independent experiments). * p < 0.05 compared with CYS9 group.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: Generated, Fluorescence

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: A, B, C, D: Optical images at 24-hour exposure. (A) Control (unexposed H9c2 cells), (B) H9c2 cells exposed to 250 μM CY, (C) H9c2 cells exposed to CYS9, and (D) H9c2 cells exposed to CYS9 presence of 1 mM NAC. Magnification, 100×. Bar = 200 μm. E, F, G, H: Induction of apoptosis in H9c2 cells by CYS9 with or without NAC. Living cell nucleii stained by Hoechst 33342 are blue. Apoptotic cells stained by FITC-conjugated probes are green. (E) Control (unexposed H9c2 cells), (F) H9c2 cells exposed for 2 hours to 250 μM CY, (G) H9c2 cells exposed to CYS9—green dots indicate apoptotic cells. (H) H9c2 cells exposed to CYS9 with 1 mM of NAC. Magnification, 100×. Bar = 200 μm.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: Control, Staining

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: Effects of NAC on reduced glutathione (GSH) levels in H9c2 cells exposed to CYS9 for 2 hours. (mean + SD from 3 independent experiments). * p < 0.01 compared with control group; § p < 0.01 compared with CYS9 group.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: Control

Journal: PLoS ONE

Article Title: Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

doi: 10.1371/journal.pone.0131394

Figure Lengend Snippet: H9c2 cell viability after (A) 24-hour and (B) 48-hour exposure to acrolein (Acr) with or without NAC was assessed by MTT assay (mean + SD from 2 independent experiments conducted in duplicate). * p < 0.05 compared with control group. † p < 0.05 compared with acrolein 100 μM group.

Article Snippet: H9c2 embryonic rat cardiomyocytes cell line and HL-60 human promyelocytic leukemia cell line specimens were purchased from American Type Culture Collection (ATCC: Manassas, VA).

Techniques: MTT Assay, Control