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: 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: 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

Journal: Asian Journal of Pharmaceutical Sciences

Article Title: High-dose treatment of cathepsin B-activatable doxorubicin prodrug nanoparticles that induce tumor-specific immunogenic cell death for immunotherapy of melanoma with minimal systemic toxicity

doi: 10.1016/j.ajps.2026.101123

Figure Lengend Snippet: Cancer cell-specific activation of CatB-NPs and induction of ICD in immune-cold melanoma cells. (A) Western blot analysis and quantification of cathepsin B expression in B16F10 cancer cells and H9C2 normal cells; (B) Confocal fluorescence images of B16F10 and H9C2 cells treated with free DOX or CatB-NPs (1 µM DOX equivalent) for 24 h, with or without the cathepsin B inhibitor Z-FA-FMK (blue: DAPI, red: DOX); (C, D) Cell viability of B16F10 cancer cells and H9C2 normal cells after 24 h treatment with free DOX or CatB-NPs; (E) Confocal microscopy and flow cytometry analyses of CRT exposure in B16F10 and H9C2 cells after 24 h treatment with PBS, DOX or CatB-NPs (red: CRT, blue: DAPI); (F, G) Quantification of ATP release and HMGB1 secretion in B16F10 and H9C2 cells treated with PBS, DOX or CatB-NPs for 24 h; (H) Confocal images of BMDMs co-cultured with B16F10 cells pretreated with DOX or CatB-NPs (red: B16F10 cells, green: BMDMs); (I) Flow cytometry of mature DCs (CD11c⁺CD40⁺CD86⁺) after co-culture with pretreated B16F10 cells.

Article Snippet: Murine melanoma B16F10 cells and rat cardiomyocyte H9C2 cells were obtained from the Korean Cell Line Bank and maintained in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone), 1% streptomycin, and 100 U/ml penicillin at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Activation Assay, Western Blot, Expressing, Fluorescence, Confocal Microscopy, Flow Cytometry, Cell Culture, Co-Culture Assay