Journal: Open Life Sciences

Article Title: IL-33/soluble ST2 axis is associated with radiation-induced cardiac injury

doi: 10.1515/biol-2022-0841

Figure Lengend Snippet: TCF7L2, Il-33, and sST2 are dysregulated in different doses of radiation-treated cardiomyocytes. After H9C2 cells were treated with 0, 2, 4, and 8 Gy X-rays for 48 h, (a) TCF7L2, (b) IL-33, and (c) sST2 levels were detected using qRT-PCR. (d) TCF7L2, IL-33, and sST2 levels in each group were examined using western blotting. *** P < 0.001; ** P < 0.01; * P < 0.05; ns: no significance.

Article Snippet: LDH activity in H9C2 cells were measured using a LDH activity assay kit (Elabscience, Wuhan, China).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Open Life Sciences

Article Title: IL-33/soluble ST2 axis is associated with radiation-induced cardiac injury

doi: 10.1515/biol-2022-0841

Figure Lengend Snippet: TCF7L2, Il-33, and sST2 are dysregulated in radiation-treated cardiomyocytes at different times. After H9C2 cells were treated with 8 Gy X-ray for 0, 12, 24, and 48 h, (a) TCF7L2, (b) IL-33, and (c) sST2 expression was detected using qRT-PCR. (d) Western blotting was performed to measure TCF7L2, IL-33, and sST2 levels in each group *** P < 0.001; ** P < 0.01; * P < 0.05; ns: no significance.

Article Snippet: LDH activity in H9C2 cells were measured using a LDH activity assay kit (Elabscience, Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Open Life Sciences

Article Title: IL-33/soluble ST2 axis is associated with radiation-induced cardiac injury

doi: 10.1515/biol-2022-0841

Figure Lengend Snippet: Knockdown of TCF7L2 decreases IL-33 and sST2 expression in radiation-induced cardiomyocytes. (a) and (b) TCF7L2 expression was measured in H9C2 cells after sh-TCF7L2 and sh-NC transfection. (c) IL-33 and (d) sST2 levels were measured using qRT-PCR after cell transfection and 8 Gy X-ray treatment for 48 h. (e) Western blotting was conducted to assess IL-33 and sST2 protein levels. *** P < 0.001; ** P < 0.01.

Article Snippet: LDH activity in H9C2 cells were measured using a LDH activity assay kit (Elabscience, Wuhan, China).

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Western Blot

Journal: Open Life Sciences

Article Title: IL-33/soluble ST2 axis is associated with radiation-induced cardiac injury

doi: 10.1515/biol-2022-0841

Figure Lengend Snippet: Mutation of IL33 promoter attenuates cell damage. H9C2 cells with wild-type and mutant IL33 promoter were treated with 0 or 8 Gy X-ray, and cell injury was assessed by detecting LDH activity. *** P < 0.001; ns, no significant.

Article Snippet: LDH activity in H9C2 cells were measured using a LDH activity assay kit (Elabscience, Wuhan, China).

Techniques: Mutagenesis, Activity 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: Biochemistry and Biophysics Reports

Article Title: Diallyl disulfide ameliorates isoproterenol induced cardiac hypertrophy activating mitochondrial biogenesis via eNOS-Nrf2-Tfam pathway in rats

doi: 10.1016/j.bbrep.2015.11.008

Figure Lengend Snippet: Effect of garlic, DADS and NaHS on cellular nitric oxide level and mitochondrial biogenesis against isoproterenol treated H9C2 cells and its inhibition by L-NAME. (A) Nitric oxide level. (B) CO1 mRNA expression. (C) Cyb mRNA expression. Data were shown as mean±SEM ( N =3), * p <0.05 versus Con, # p <0.05 versus Iso.

Article Snippet: H9C2 cells were purchased from Lonza, USA and cultured using DMEM media containing10% fetal bovine serum (FBS).

Techniques: Inhibition, Expressing

Journal: Toxics

Article Title: Transient Post-Natal Exposure to Xenoestrogens Induces Long-Term Alterations in Cardiac Calcium Signaling

doi: 10.3390/toxics10030102

Figure Lengend Snippet: Estradiol benzoate influences SERCA2 expression in cardiomyocytes. H9C2 cells plated in 6-well plates were treated for 72 h with estradiol benzoate at 10 nM, 100 nM, and 1000 nM, or ethanol (EB0). In these cells, SERCA2 protein levels were measured via Western blot. β-ACTIN was used as loading control. Data are expressed as mean ± SEM; n = 3 per group. ANOVA with a Fisher’s LSD test was performed to compare control and EB-treated cells. * p < 0.05. The results are representative of two independent experiments.

Article Snippet: The H9C2 cell line, estradiol benzoate, the polyvinylidene difluoride (PVDF) membrane, the RIPA buffer and the anti-β-ACTIN antibody were acquired at Merck KGaA (Darmstadt, Germany).

Techniques: Expressing, Western Blot, Control

Journal: Toxics

Article Title: Transient Post-Natal Exposure to Xenoestrogens Induces Long-Term Alterations in Cardiac Calcium Signaling

doi: 10.3390/toxics10030102

Figure Lengend Snippet: Effects of estradiol benzoate on oxidative stress in cardiomyocytes. H9C2 cells grown on coverslips were incubated with estradiol benzoate at 100 nM and 1000 nM or ethanol (EB0) for 72 h. ( A ) Cells grown on coverslips were incubated with an antibody targeting 8-OHdG (green) (Scale bar = 100 μm). ( B ) In these cells, antioxidant enzymes (Superoxide dismutase 1 and 2, SOD1, SOD2) and the regulator of mitochondrial oxidative phosphorylation COX2 were measured via Western blot. GAPDH protein levels were used as loading control. Data are expressed as mean ± SEM; n = 4 per group. ANOVA with a Fisher’s LSD test was performed to compare control and EB-treated cells. ns: not statistically significant.

Article Snippet: The H9C2 cell line, estradiol benzoate, the polyvinylidene difluoride (PVDF) membrane, the RIPA buffer and the anti-β-ACTIN antibody were acquired at Merck KGaA (Darmstadt, Germany).

Techniques: Incubation, Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Apelin receptor inhibition in ischemia-reperfused mouse hearts protected by endogenous n-3 polyunsaturated fatty acids

doi: 10.3389/fphar.2023.1145413

Figure Lengend Snippet: Expression of intracellular protein kinases in EPA-treated H9C2 cells under simulated IR conditions. (A) EPA treatment protocol. Cells were deprived of oxygen for 24 h, then EPA was added and reoxygenated for 6 h. EPA treatment improved morphology of the cell subjected to HR (B) . Protein expression of APLNR was inhibited in hypoxia-reoxygenation (HR) cells after treatment with EPA (C,D) . EPA treatment alters the activation of the PI3K-AKT-mTOR signaling axis, as displayed in the representative images (C) and quantitative analysis of expression of phosphorylated and total PI3K (E) , AKT (F) , and mTOR (G) . n = 6 mice per group. (H–K) Representative images and quantitative analysis of expression of PKCδ (I) , PKCε (J) , and phosphorylated (p-) and total (t-) p38α (K) . n = 6 mice per group. Data are shown as the mean ± SEM; * p < 0.05 vs. Con (blue) or HR (green); NS, not significant compared with Con (blue) or HR (green).

Article Snippet: H9C2 cells (iCell-r012) were employed to assess the expression of APLNR and intracellular phosphorylation signaling components following hypoxia and reoxygenation (principal components of myocardial IR).

Techniques: Expressing, Activation Assay

Journal: PLoS ONE

Article Title: A Non-Neuronal Cardiac Cholinergic System Plays a Protective Role in Myocardium Salvage during Ischemic Insults

doi: 10.1371/journal.pone.0050761

Figure Lengend Snippet: (A) Stably transfected ChAT KO cells with reduced β-catenin immunoreactivity (signal intensity: 80.7±4.7% vs. control, P <0.05; rate with the linear form: 20.1±2.2% vs. control, P <0.05) observed between cells (A−b) were easily separated by simple agitation without forming large aggregates as compared with control LacZ KO cells (A−a). The intensities and lengths of β-catenin signals with linear forms were measured using Image J, and the rate of cells expressing strong β-catenin signals in the intercellular spaces were calculated (A−b). ChAT KO cells exhibited fewer ACh immunoreactive signals at the cell membrane (A−c). Representative data from 5 independent experiments were shown. Scale bar: 10 µm. (B) ChAT KO HL-1 cells (murine cardiomyocyte-derived HL-1 cells stably transfected with ChAT specific miR RNAi expression vector) expressed less Cx43 compared with control LacZ KO HL-1 cells, as shown by western blot (Cx43: 48.7±5.0% vs. control, P <0.01) (B−a) and immunocytochemistry (B−b). The Cx43 signals in cytoplasm and intercellular spaces were attenuated in ChAT KO HL-1 cells compared with control cells (signal intensity: 41.6±5.1%, P <0.05; signal length: 25.8±1.5%, P <0.05) (B−b). The signal intensities and lengths of Cx43 signals were evaluated by Image J. Representative data from 5 independent experiments were shown. Scale bar: 10 µm. LY dye transfer, initiated by scratching cells and thereafter stained with LY dye, was suppressed in linearly aligned ChAT KO HL-1 cells, as compared with control cells (vs. control: 28.0±4.0%; P <0.01, n = 10) (B−c). The non-neuronal cholinergic system in cardiomyocytes played a role in maintaining cell−cell communication via gap junction. Scale bar: 10 µm. (C) ChAT KO HEK cells with a few ACh (vs. control: 9.3±3.5%; P <0.01, n = 9; C−a) reciprocally increased MTT activity (vs. control: 384.8±20.8%; P <0.01, n = 9; C−b) and consumed more oxygen (vs. control: −239.4±26.4%; P <0.01, n = 9; C−c). (D) ChAT KO HEK cells also showed reciprocally lower ATP levels than control cells (control vs. ChAT KO: 765.1±56.9 vs. 425.7±18.7 mM/g protein; P <0.01, n = 6). (E) After inducing chemical hypoxia with 2 mM of CoCl 2 more ChAT KO HEK cells died compared with control cells (vs. control: 26.6±2.33%; P <0.01, n = 7). (F) Similarly, ChAT KO HL-1 cardiac cells also consumed more ATP than control cells (control vs. ChAT KO: 271.9±28.2 vs. 83.1±8.8 mM/g protein; P <0.01, n = 7), indicating that the non-neuronal cardiac cholinergic system inhibited energy wasting. (G) Similar to the ChAT gene knockdown, the ChAT inhibitor HC-3 (10 µM) also increased MTT activities (G−a) and ATP consumption in rat myocardium-derived H9c2 cells ( P <0.05, n = 4; G−b).

Article Snippet: HEK293 cells and H9c2 cells, which were spontaneously immortalized ventricular myoblasts from rat embryos, purchased from DS Pharma Biomedical, were cultured in DMEM (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% FBS and antibiotics.

Techniques: Stable Transfection, Transfection, Control, Expressing, Membrane, Derivative Assay, Plasmid Preparation, Western Blot, Immunocytochemistry, Staining, Activity Assay, Knockdown