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: Journal of Cardiothoracic Surgery

Article Title: Inhibition of mir-155-5p alleviates cardiomyocyte pyroptosis induced by hypoxia/reoxygenation via targeting SIRT1-mediated activation of the NLRP3 inflammasome

doi: 10.1186/s13019-025-03366-1

Figure Lengend Snippet: H/R induced NLRP3-mediated pyroptosis in H9c2 cells. The H9c2 cells in the H/R group were subjected to hypoxia for 6 h and reoxygenation for 4 h, while normal cultured H9c2 cells were designated as the Control group. ( A ) The proliferative activity of H9c2 cells was assessed using the CCK-8 assay. ( B ) The level of LDH released by H9c2 cells was quantified using colorimetric analysis. ( C ) The death of H9c2 cells was observed by Calcein-AM/PI staining. The live cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. The levels of IL-1β ( D ) and IL-18 ( E ) in the supernatant of H9c2 cell were detected by ELISA. ( F ) The protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells were assessed using Western blot. n = 3. ** P < 0.01

Article Snippet: Rat cardiomyocytes H9c2 were purchased from the Cell Bank of China Center for Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Mediums (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C, 5% CO 2 .

Techniques: Cell Culture, Control, Activity Assay, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Journal of Cardiothoracic Surgery

Article Title: Inhibition of mir-155-5p alleviates cardiomyocyte pyroptosis induced by hypoxia/reoxygenation via targeting SIRT1-mediated activation of the NLRP3 inflammasome

doi: 10.1186/s13019-025-03366-1

Figure Lengend Snippet: Inhibition of miR-155-5p attenuates NLRP3 inflammasome mediated pyroptosis in H/R-exposed H9c2 cells. ( A ) The expression level of miR-155-5p in H9c2 cells was quantified using qRT-PCR. The miR-155-5p inhibitor and the inhibitor-NC were separately transfected into H9c2 cells, followed by exposure to H/R. ( B ) The expression of miR-155-5p in H9c2 cells was assessed by qRT-PCR following transfection with a miR-155-5p inhibitor and inhibitor-NC. ( C ) CCK-8 assay was used to detect the proliferative activity of H9c2 cells. ( D ) Colorimetric analysis was used to quantify LDH activity in the supernatant of H9c2 cells. ( E ) Calcein-AM /PI staining was used to observe the death of H9c2 cells. The viable cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. ( F - G ) ELISA was used to determine the levels of IL-1β and IL-18 in the supernatant of H9c2 cells. ( H ) Western blot was used to assess the protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells. n = 3. * P < 0.05, ** P < 0.01

Article Snippet: Rat cardiomyocytes H9c2 were purchased from the Cell Bank of China Center for Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Mediums (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C, 5% CO 2 .

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transfection, CCK-8 Assay, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Cardiothoracic Surgery

Article Title: Inhibition of mir-155-5p alleviates cardiomyocyte pyroptosis induced by hypoxia/reoxygenation via targeting SIRT1-mediated activation of the NLRP3 inflammasome

doi: 10.1186/s13019-025-03366-1

Figure Lengend Snippet: SIRT1 overexpression inhibits the activation of NLRP3 inflammasome in H/R-induced H9c2 cells. ( A ) The mRNA expression level of SIRT1 in H9c2 cells was detected by qRT-PCR. ( B ) The protein expression level of SIRT1 in H9c2 cells was assessed by Western blot. The SIRT1 overexpression plasmid (oe-SIRT1) and empty carrier plasmid (Vector) were separately transfected into H9c2 cells, followed by exposure to H/R. The expression levels of SIRT1 mRNA ( C ) and protein ( D ) in H9c2 cells were quantified using qRT-PCR and Western blot. ( E ) The proliferative activity of H9c2 cells was assessed using the CCK-8 assay. ( F ) The LDH activity in the supernatant of H9c2 cells was quantified using colorimetric analysis. ( G ) The death of H9c2 cells was observed by Calcein-AM /PI staining. The viable cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. ( H - I ) The levels of IL-1β and IL-18 in the supernatant of H9c2 cells were detected by ELISA. ( J ) The protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells were assessed using Western blot. n = 3. * P < 0.05, ** P < 0.01

Article Snippet: Rat cardiomyocytes H9c2 were purchased from the Cell Bank of China Center for Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Mediums (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C, 5% CO 2 .

Techniques: Over Expression, Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Transfection, Activity Assay, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiothoracic Surgery

Article Title: Inhibition of mir-155-5p alleviates cardiomyocyte pyroptosis induced by hypoxia/reoxygenation via targeting SIRT1-mediated activation of the NLRP3 inflammasome

doi: 10.1186/s13019-025-03366-1

Figure Lengend Snippet: miR-155-5p directly targets SIRT1 in H9c2 cells. ( A ) The binding sites between miR-155-5p and SIRT1 were predicted. ( B ) The relative fluorescence activity of SIRT1-WT and SIRT1-MUT in H9c2 cells was assessed using a dual luciferase assay. The H9c2 cells were transfected with the miR-155-5p inhibitor and the inhibitor-NC, respectively. ( C ) The expression level of SIRT1 mRNA in H9c2 cells was determined by qRT-PCR. ( D ) The protein expression level of SIRT1 in H9ce cells was detected by Western blot. n = 3. ** P < 0.01

Article Snippet: Rat cardiomyocytes H9c2 were purchased from the Cell Bank of China Center for Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Mediums (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C, 5% CO 2 .

Techniques: Binding Assay, Fluorescence, Activity Assay, Luciferase, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Cardiothoracic Surgery

Article Title: Inhibition of mir-155-5p alleviates cardiomyocyte pyroptosis induced by hypoxia/reoxygenation via targeting SIRT1-mediated activation of the NLRP3 inflammasome

doi: 10.1186/s13019-025-03366-1

Figure Lengend Snippet: SIRT1 silencing partially reversed the inhibition of H/R-induced H9c2 cell pyroptosis by miR-155-5p inhibitor. The miR-155-5p inhibitor and SIRT1 siRNA interference plasmid (si-SIRT1) or negative control plasmid (si-NC) were transfected into H9c2 cells, followed by exposure to H/R. ( A ) Western blot was used to detect the protein expression level of SIRT1 in H9c2 cells. ( B ) CCK-8 assay was used to assess the proliferative activity of H9c2 cells. ( C ) Colorimetric analysis was used to quantify LDH activity in the supernatant of H9c2 cells. ( D ) Calcein-AM /PI staining was used to observe the death of H9c2 cells. The viable cells were stained green by Calcein-AM, whereas the dead cells were stained red by PI. Scale bar = 50 μm. ( E - F ) ELISA was used to determine the levels of IL-1β and IL-18 in the supernatant of H9c2 cells. ( G ) Western blot was used to assess the protein expression levels of NLRP3, GSDMD-N, and C-caspase-1 in H9c2 cells. n = 3. * P < 0.05, ** P < 0.01

Article Snippet: Rat cardiomyocytes H9c2 were purchased from the Cell Bank of China Center for Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Mediums (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C, 5% CO 2 .

Techniques: Inhibition, Plasmid Preparation, Negative Control, Transfection, Western Blot, Expressing, CCK-8 Assay, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cardiovascular Therapeutics

Article Title: Long Noncoding RNA SNHG4 Attenuates the Injury of Myocardial Infarction via Regulating miR-148b-3p/DUSP1 Axis

doi: 10.1155/2022/1652315

Figure Lengend Snippet: Overexpression of SNHG4 promotes the viability and represses the apoptosis and inflammation of hypoxia-induced H9c2 cells. (a) The expression of SNHG4 in hypoxia-induced H9c2 cells was detected by qRT-PCR. (b) The expression of SNHG4 in hypoxia-induced H9c2 cells transfected with pcDNA-SNHG4/pcDNA-NC was detected by qRT-PCR. (c) Cell viability (OD450) was measured by MTT assay. (d) Caspase-3 activity. (e) The protein expression of Bax and Bcl-2 were measured by western blot. (f–h) The levels of TNF- α , IL-6, and IL-1 β were measured by ELISA. ∗∗ P < 0.01.

Article Snippet: A rat myocardial cell line H9c2 (BioVector, Beijing, China) was cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, MTT Assay, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cardiovascular Therapeutics

Article Title: Long Noncoding RNA SNHG4 Attenuates the Injury of Myocardial Infarction via Regulating miR-148b-3p/DUSP1 Axis

doi: 10.1155/2022/1652315

Figure Lengend Snippet: The target relationship between miR-148b-3p and SNHG4. (a) A binding site of SNHG4 and miR-148b-3p was predicted by StarBase. (b) DLR assay of the target relationship between miR-148b-3p and SNHG4. (c) The expression of SNHG4 in H9c2 cells transfected with sh-SNHG4/sh-NC was detected by qRT-PCR. (d) The expression of miR-148b-3p in H9c2 cells transfected with pcDNA-SNHG4/pcDNA-NC/sh-SNHG4/sh-NC was detected by qRT-PCR. ∗∗ P < 0.01.

Article Snippet: A rat myocardial cell line H9c2 (BioVector, Beijing, China) was cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Binding Assay, Expressing, Transfection, Quantitative RT-PCR

Journal: Cardiovascular Therapeutics

Article Title: Long Noncoding RNA SNHG4 Attenuates the Injury of Myocardial Infarction via Regulating miR-148b-3p/DUSP1 Axis

doi: 10.1155/2022/1652315

Figure Lengend Snippet: Silencing of miR-148b-3p promotes the viability and represses the apoptosis and inflammation of hypoxia-induced H9c2 cells. (a) The expression of miR-148b-3p in hypoxia-induced H9c2 cells was detected by qRT-PCR. (b) The expression of miR-148b-3p in hypoxia-induced H9c2 cells transfected with miR-148b-3p inhibitor/inhibitor-NC was detected by qRT-PCR. (c) Cell viability (OD450) was measured by MTT assay. (d) Caspase-3 activity. (e) The protein expression of Bax and Bcl-2 were measured by western blot. (f–h) The levels of TNF- α , IL-6, and IL-1 β were measured by ELISA. ∗∗ P < 0.01.

Article Snippet: A rat myocardial cell line H9c2 (BioVector, Beijing, China) was cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Transfection, MTT Assay, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cardiovascular Therapeutics

Article Title: Long Noncoding RNA SNHG4 Attenuates the Injury of Myocardial Infarction via Regulating miR-148b-3p/DUSP1 Axis

doi: 10.1155/2022/1652315

Figure Lengend Snippet: The target relationship between DUSP1 and miR-148b-3p. (a) A binding site of DUSP1 and miR-148b-3p was predicted by TargetScan and StarBase. (b) DLR assay of the target relationship between DUSP1 and miR-148b-3p. (c) The expression of miR-148b-3p in H9c2 cells transfected with miR-148b-3p mimics/miR-NC was detected by qRT-PCR. (d) The protein expression of DUSP1 in H9c2 cells transfected with miR-148b-3p inhibitor/inhibitor NC or miR-148b-3p mimics/miR-NC was detected by western blot. (e) The mRNA expression of DUSP1 in hypoxia-induced H9c2 cells was detected by qRT-PCR. (f) The protein expression of DUSP1 in hypoxia-induced H9c2 cells was detected by western blot. (g) The mRNA expression of DUSP1 in hypoxia-induced H9c2 cells transfected with sh-DUSP1/sh-NC/pcDNA-NC/pcDNA-DUSP1 was detected by qRT-PCR. (h) Cell apoptosis was detected by flow cytometry. ∗∗ P < 0.01.

Article Snippet: A rat myocardial cell line H9c2 (BioVector, Beijing, China) was cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Binding Assay, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Cardiovascular Therapeutics

Article Title: Long Noncoding RNA SNHG4 Attenuates the Injury of Myocardial Infarction via Regulating miR-148b-3p/DUSP1 Axis

doi: 10.1155/2022/1652315

Figure Lengend Snippet: The regulatory mechanisms of SNHG4 involving miR-148b-3p/DUSP1 in hypoxia-induced H9c2 cells. (a) The protein expression of DUSP1 in hypoxia-induced H9c2 cells was determined by western blot. (b) Cell viability (OD450) was measured by MTT assay. (c) Caspase-3 activity. (d) The protein expression of Bax and Bcl-2 were measured by western blot. (e–g) The levels of TNF- α , IL-6, and IL-1 β were measured by ELISA. ∗ P < 0.05; ∗∗ P < 0.01.

Article Snippet: A rat myocardial cell line H9c2 (BioVector, Beijing, China) was cultured in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, MTT Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

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: Oncotarget

Article Title: Over-expression of growth differentiation factor 15 (GDF15) preventing cold ischemia reperfusion (I/R) injury in heart transplantation through Foxo3a signaling

doi: 10.18632/oncotarget.16607

Figure Lengend Snippet: H9C2 cells (80,000 cells/well) were plated in six well plates and allowed to culture at 37°C 5% CO 2 overnight. Cells were infected with human GDF15 expression adenovirus or control null virus for 24h. Cells were then subjected to a hypoxia chamber with 0% O 2 and 15% CO 2 at 10°C for 16 h, followed by 24h reperfusion at 5% CO 2 , 28% O 2 at 37°C. Gene expression of GDF15 was detected by RT-PCR and cell apoptosis was detected by double staining with FITC labeled Annexin-V and PI and flow cytometry. (A) Expression of human GDF15. Human GDF15 expression in H9C2 cells was detected by RT-PCR using primers specific to human GDF15 sequence and Western blotting. Left panel: regular RT-PCR; Middle panel: qRT-PCR. Right panel; Western blotting. (B) Cell apoptosis/death detected by Annenix-V and PI binding. Upper panel: representative data; bottom panel: Summarized data of flow cytometry for cell apoptosis/death. (C) Representative images of cell death detected by an incucyte system. (D) A cell death curve over time. Data are the summary of three independent experiments. * p< 0.05 was defined as statistical significance.

Article Snippet: Rat heart cell line H9C2 were purchased from ATCC (Manassas, VA 20108), cultured and maintained in DMEM medium (Invitrogen, Canada) which were supplemented with 10% fetal bovine serum (Sigma, Oakville, ON, Canada) and 100 U penicillin and streptomycin.

Techniques: Infection, Expressing, Control, Virus, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Double Staining, Labeling, Flow Cytometry, Sequencing, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: Oncotarget

Article Title: Over-expression of growth differentiation factor 15 (GDF15) preventing cold ischemia reperfusion (I/R) injury in heart transplantation through Foxo3a signaling

doi: 10.18632/oncotarget.16607

Figure Lengend Snippet: (A) Phosphorylation of Foxo3a in the heart grafts. Total protein was extracted from heart grafts at day 3 post transplantation (n=6). Phosphorylation of Foxo3a was detected by Western blotting with primary antibodies against phosphorylated Foxo3a. Left panel: Representative images of Western blotting for Foxo3a. Right panel: Densitometry values of p-Foxo3a/β-actin. (B) Over expression of GDF15 recovered expression of phosphorylated Foxo3a in vitro . H9C2 cells (80,000 cells/well) were plated in six well plates and allowed to culture at 37°C 5% CO 2 overnight. Cells were infected with human GDF15 expression adenovirus or control null virus, for 24 h, Cells were then subjected to a hypoxia chamber with 0% O 2 and 15% CO 2 at 10°C for 16 h, followed by a 24 h reperfusion period at 5% CO 2 , 28% O 2 at 37°C. Total protein was extracted from the cells and the expression of GDF15, p-Foxo3a and β-actin were detected by Western blotting using appropriate antibodies. Left panel: Representative images of Western blotting for p-Foxo3a, and β-actin from three independent experiments. Right panel Densitometry values of p-Foxo3a/β-actin. (C) GDF15 siRNA reduced GDF15 expression. H9C2 cells were transfected with GDF15 siRNA prior to hypoxia/reperfusion. Left panel: Representative images of Western blotting for GDF15 and β-actin from three independent experiments. Right panel Densitometry values of GDF15/β-actin. (D) GDF15 siRNA reduced phosphorylated Foxo3a expression. Left panel: Representative images of Western blotting for p-Foxo3a, and β-actin from three independent experiments. Right panel Densitometry values of p-Foxo3a/β-actin* p< 0.05 was defined as statistical significance.

Article Snippet: Rat heart cell line H9C2 were purchased from ATCC (Manassas, VA 20108), cultured and maintained in DMEM medium (Invitrogen, Canada) which were supplemented with 10% fetal bovine serum (Sigma, Oakville, ON, Canada) and 100 U penicillin and streptomycin.

Techniques: Phospho-proteomics, Transplantation Assay, Western Blot, Over Expression, Expressing, In Vitro, Infection, Control, Virus, Transfection

Journal: Pharmaceuticals

Article Title: DHDK, a Plant-Derived Natural Small Molecule, Protects Against Doxorubicin-Induced Cardiotoxicity via the PPARG-CPT1B-FAO Axis

doi: 10.3390/ph18111759

Figure Lengend Snippet: Integrated network toxicology analysis of DOX targets in cardiotoxicity and chronic disorders. CCK-8 results confirm that DHDK can alleviate cardiomyocyte damage caused by DOX ( a ), Venn diagram of DOX and potential targets of cardiotoxicity ( b ), Venn diagrams of DOX and cardiotoxicity, potential targets of cardiac aging ( c ), and Venn diagrams of potential targets of cardiotoxicity caused by DOX and cardiac aging and lipid metabolism ( d ). Protein–protein Interaction (PPI) plot ( e ), Gene Ontology (GO) results ( f – h ), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis ( i ) results of DOX-induced cardiotoxicity and cardiac aging. Data are presented as mean ± SD ( n = 3). Statistical significance was determined by Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns indicated no statistical significance.

Article Snippet: H9c2 rat cardiomyocytes and the CCK-8 kit (G4103) were purchased from Servicebio (Wuhan, China).

Techniques: CCK-8 Assay

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