Journal: Frontiers in Endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: Screening of a novel therapeutic target for DbCM. (A) Relative mRNA expression of hub genes normalized to a GAPDH internal control (n=6 each). Values are expressed relative to control groups. (B, C) Representative Western blots and analysis of ANGPTL4 expression; p-FAK(Y397)/FAK ratio; SIRT3 expression; Ac-SOD2/SOD2 ratio; P67phox expression; Gp91phox expression; P47phox expression; Cleaved caspase-3 expression; Bcl-2 expression; Bax expression. Equal protein loading was confirmed using an anti-GAPDH antibody (n=6 each). (D) Representative images of ANGPTL4 fluorescence in heart sections. Scale bar, 50 μm. (E) Representative images of DHE fluorescence in heart sections. Scale bar, 50 μm. (F) Representative images of apoptotic cardiomyocytes (200×). The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 μm. (G) Representative immunohistochemical stainings of ANGPTL4, P47phox, and Cleaved caspase-3 (n=6 each). Scale bars, 50 μm. (H) Quantification of ANGPTL4 fluorescence intensity in heart sections (n=6 each). (I) Quantification of DHE fluorescence intensity in heart sections (n=6). (J) Percentage of TUNEL positive cells. (K–M) Representative immunohistochemical quantification of ANGPTL4, P47phox, and Cleaved caspase-3. Scale bars, 50 μm. Data are shown as mean ± SD (n=6 each); * p < 0.05, ** p < 0.01 vs CON group (student t -test). ns represents statically non-significant; IOD, the integral optical density.

Article Snippet: Human cardiomyocytes (AC16) were purchased from Sciencell (Cat# 6200, San Diego, CA, USA).

Techniques: Expressing, Western Blot, Fluorescence, TUNEL Assay, Immunohistochemical staining

Journal: Frontiers in Endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: ANGPTL4 siRNA exhibited the anti-apoptotic and anti-oxidative effects in HG-treated AC16 cardiomyocytes. (A) Representative Western blots showing ANGPTL4, p-FAK(Y397), FAK, and SIRT3 levels in AC16 cardiomyocytes at indicated time points after treatment with HG (30 mM). (B) WB analysis of ANGPTL4 expression, p-FAK(Y397)/FAK ratio, and SIRT3 expression at indicated time points, respectively. (C, D) Representative Western blots and quantification of p-FAK(Y397), FAK, SIRT3, Ac-SOD2, SOD2, P67phox, P47phox, Cleaved caspase-3, Bcl-2, and Bax in cultured AC16 cardiomyocytes under the control or HG (30 mM) condition plus treatment with si-RNA targeting ANGPTL4 (si-ANGPTL4) or negative control siRNA (si-NC). Equal protein loading was confirmed using an anti-β-tubulin antibody. (E, G) Representative images and quantification of DHE fluorescence in cultured AC16 cardiomyocytes under the control or HG (30 mM) condition plus treatment with si-RNA targeting ANGPTL4 (si-ANGPTL4) or negative control siRNA (si-NC). Scale bars, 50 μm. (F, H) Representative images of apoptotic cardiomyocytes (200×) and percentage of TUNEL positive cells. The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 μm. Experiments were repeated at least three times independently with similar results. * p < 0.05, ** p < 0.01 vs CON group. # p < 0.05 vs the CON+SiNC group; & p < 0.05 vs the HG+SiNC group; $ p < 0.05 vs the HG+SiNC group (student t -test).

Article Snippet: Human cardiomyocytes (AC16) were purchased from Sciencell (Cat# 6200, San Diego, CA, USA).

Techniques: Western Blot, Expressing, Cell Culture, Negative Control, Fluorescence, TUNEL Assay

Journal: Frontiers in Endocrinology

Article Title: Weighted Gene Co-Expression Network Analysis Identifies ANGPTL4 as a Key Regulator in Diabetic Cardiomyopathy via FAK/SIRT3/ROS Pathway in Cardiomyocyte

doi: 10.3389/fendo.2021.705154

Figure Lengend Snippet: ANGPTL4 elevated SOD2 acetylation, oxidative stress, and apoptosis. (A, B) Representative immunoblots showing SIRT3, p-FAK(Y397), and FAK levels in AC16 cardiomyocytes at 48 h after different concentrations of ANGPTL4 treatment. (C, D) Representative Western blots and quantification of p-FAK(Y397), FAK, P67phox, SIRT3, SOD2,Ac-SOD2, Bax, Bcl-2, and Cleaved caspase-3 in AC16 cardiomyocytes after treatment with BSA or ANGPTL4 (200 ng/ml each) for 48 h. (E, G) Representative images of apoptotic cardiomyocytes (200×) after treatment with BSA or ANGPTL4 (200 ng/ml each) for 48 h and percentage of TUNEL positive cells. The apoptotic cells were detected by TUNEL (green), and the nuclei were detected by DAPI (blue). Scale bar, 50 μm. (F, H) Representative images and quantification of DHE fluorescence in AC16 cardiomyocytes after treatment with BSA or ANGPTL4 (200 ng/ml) for 48 h. Scale bar, 50 μm. (I) Schematic illustration shows ANGPTL4 interacting with integrin, inducing apoptosis in the Diabetic Cardiomyopathy via FAK/SIRT3/ROS pathway in cardiomyocyte. P represents phosphorylation, α and β represent subunit of the integrin. Data are shown as mean ± SD; * p < 0.05, ** p < 0.01 vs BSA group (student t test).

Article Snippet: Human cardiomyocytes (AC16) were purchased from Sciencell (Cat# 6200, San Diego, CA, USA).

Techniques: Western Blot, TUNEL Assay, Fluorescence

Journal: iScience

Article Title: Cystine-cored diphenylalanine appended peptide-based self-assembled fluorescent nanostructures direct redox-responsive drug delivery

doi: 10.1016/j.isci.2024.109523

Figure Lengend Snippet:

Article Snippet: The human cardiac myocyte cell line (AC16) was procured from Merck, Millipore, USA.

Techniques: Recombinant, Protease Inhibitor, Modification, Saline, Software

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: The expression of circ_0124644 in AMI patients and hypoxia-induced AC16 cells. (A) The circ_0124644 expression in the serum of AMI patients and healthy control volunteers was measured using qRT-PCR. (B) The expression of circ_0124644 was detected using qRT-PCR in hypoxia-treated and normoxia-treated AC16 cells. RNase R assay (C) and Act D assay (D) were used to confirm the stability of circ_0124644. (E) Subcellular localization analysis was performed to assess the distribution of circ_0124644 in the cytoplasm and nuclear of AC16 cells. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: The regulation of circ_0124644 silencing on hypoxia-induced cardiomyocytes injury. (A) AC16 cells were transfected with sh-NC or sh-circ_0124644. The expression of circ_0124644 was measured by qRT-PCR. (B–I) AC16 cells were transfected with sh-NC or sh-circ_0124644, followed by treated with hypoxia. AC16 cells treated with normoxia were used as control. (B) CCK8 assay was used to examine cell viability. (C,D) Flow cytometry was performed to detect cell cycle distribution and cell apoptosis. (E) The protein levels of CyclinD1 and Cleaved-casp3 were determined using WB analysis. (F–I) Corresponding Assay Kits were used to assess the levels of LDH, MDA, SOD, and CAT. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: Circ_0124644 acted as a sponge of miR-590-3p. (A) The predicted and mutated binding sites between miR-590-3p and circ_0124644 were exhibited. (B) The transfection efficiency of miR-590-3p mimic was confirmed by detecting miR-590-3p expression in AC16 cells using qRT-PCR. Dual-luciferase reporter assay (C) , RIP assay (D) , and RNA pull-down assay (E) were employed to assess the interaction between miR-590-3p and circ_0124644. (F) AC16 cells were transfected with sh-NC or sh-circ_0124644, followed by treated with hypoxia. AC16 cells treated with normoxia were used as control. The expression of miR-590-3p was examined by qRT-PCR. (G) The expression of miR-590-3p in the serum of AMI patients and healthy control volunteers was determined by qRT-PCR. (H) Pearson correlation analysis was used to assess the correlation between miR-590-3p and circ_0124644. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

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

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: The regulation of miR-590-3p overexpression on cardiomyocytes injury induced by hypoxia. AC16 cells were transfected with miR-NC or miR-590-3p mimic, followed by treated with hypoxia. Normoxia-treated AC16 cells were used as control. (A) Cell viability was determined using CCK8 assay. (B,C) Cell cycle distribution and cell apoptosis were detected by flow cytometry. (D) WB analysis was performed to evaluate the protein levels of CyclinD1 and Cleaved-casp3. (E–H) The levels of LDH, MDA, SOD, and CAT were determined using corresponding Assay Kits. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Over Expression, Transfection, Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: Effects of miR-590-3p inhibitor and circ_0124644 silencing on hypoxia-induced cardiomyocytes injury. (A) AC16 cells were transfected with anti-NC or anti-miR-590-3p. MiR-590-3p expression was detected by qRT-PCR. (B–I) AC16 cells were transfected with sh-NC + anti-NC, sh-circ_0124644 + anti-NC, sh-circ_0124644 + anti-miR-590-3p, followed by treated with hypoxia. Normoxia-treated AC16 cells transfected with sh-NC + anti-NC were used as control. (B) CCK8 assay was employed to measure cell viability. (C,D) Cell cycle distribution and cell apoptosis were evaluated using flow cytometry. (E) The protein levels of CyclinD1 and Cleaved-casp3 were examined by WB analysis. (F–I) The levels of LDH, MDA, SOD, and CAT were analyzed by corresponding Assay Kits. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Control, CCK-8 Assay, Flow Cytometry

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: SOX4 could be targeted by miR-590-3p. (A) The predicted and mutated binding sites between SOX4 3′UTR and miR-590-3p were shown. (B) Dual-luciferase reporter assay was used to confirm the interaction between SOX4 and miR-590-3p. (C) AC16 cells were transfected with miR-NC or miR-590-3p mimic, followed by treated with hypoxia. AC16 cells treated with normoxia were used as control. The protein expression of SOX4 was analyzed using WB analysis. (D) AC16 cells were transfected with sh-NC + anti-NC, sh-circ_0124644 + anti-NC, sh-circ_0124644 + anti-miR-590-3p, followed by treated with hypoxia. Normoxia-treated AC16 cells transfected with sh-NC + anti-NC were used as control. WB analysis was used to detect SOX4 protein expression. (E,F) The protein and mRNA expression levels of SOX4 in the serum of AMI patients and healthy control volunteers were evaluated using WB analysis and qRT-PCR. (G,H) The correlation between SOX4 and miR-590-3p or circ_0124644 was assessed by Pearson correlation analysis. ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

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

Journal: Frontiers in Genetics

Article Title: Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4

doi: 10.3389/fgene.2021.667724

Figure Lengend Snippet: Effects of SOX4 and miR-590-3p overexpression on hypoxia-induced cardiomyocytes injury. (A) WB analysis was used to detect SOX4 protein expression in AC16 cells to evaluate the transfection efficiency of pcDNA SOX4 overexpression vector. (B–I) AC16 cells were transfected with miR-NC + pcDNA, miR-590-3p + pcDNA, or miR-590-3p + SOX4, followed by treated with hypoxia. Normoxia-treated AC16 cells transfected with miR-NC + pcDNA were used as control. (B) Cell viability was detected by CCK8 assay. (C,D) Flow cytometry was utilized for assessing cell cycle distribution and cell apoptosis. (E) WB analysis was used to test the protein levels of CyclinD1 and Cleaved-casp3. (F–I) Corresponding Assay Kits were employed to detect the levels of LDH, MDA, SOD, and CAT. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human cardiomyocytes (AC16) (Biovector National Typical Culture Center, Beijing, China) were cultured in DMEM medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States).

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Flow Cytometry

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Dexmedetomidine (DEX) has a protective effect on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. (A) Cell -Counting Kit 8 (CCK-8) was used to measure cell viability. (B, C) Apoptosis and apoptotic rate were determined using flow cytometry. (D) GSE126104 showed that DEX decreased ectodysplasin-A2 receptor (EDA2R) expression in the left ventricle of rats. ***, P <0.001 vs. con. (E) The expression of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. ** P <0.01; *** P <0.001; **** P <0.0001 vs. con. ## P <0.01; #### P <0.0001 vs. H/R. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Cell Counting, CCK-8 Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown suppresses hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis. (A) The knockdown efficiency of EDA2R in cardiomyocytes was detected by RT-qPCR and western blotting. (B) Cell -Counting Kit 8 (CCK-8) was applied to detect cell viability. (C, D) Apoptosis was determined using flow cytometry, and apoptotic rate was calculated. *** P <0.001; **** P <0.0001 vs. con. ## P <0.01; ### P <0.001; #### P <0.0001 vs. H/R+shNC. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Flow Cytometry

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown represses mitochondria-mediated apoptosis. (A) Mitochondrial morphology of cardiomyocytes was observed by transmission electron microscopy (the arrows represented mitochondrial morphology, scale bar=500 nm). (B) Mitochondrial membrane potential (MMP) was detected by JC-1 (Scale bar=100 µ m, Red: **** P <0.0001 vs. con. or hypoxia/reoxygenation (H/R)+shNC; Green: #### P <0.0001 vs. con. H/R+shNC). (C, D) The levels of cytochrome C in mitochondria and cytoplasm of cardiomyocytes were determined by western blotting. (E) The expressions of Bax and Bcl-2 were determined in cardiomyocytes by western blotting. (F, G) Caspase-3 and Caspase-9 activity in cardiomyocytes was detected. *** P <0.001; **** P <0.0001 vs. con. # P <0.05; ## P <0.01; ### P <0.001; #### P <0.0001vs. H/R+shNC. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Activity Assay

Journal: Experimental Animals

Article Title: Ectodysplasin-A2 receptor (EDA2R) knockdown alleviates myocardial ischemia/reperfusion injury through inhibiting the activation of the NF-κB signaling pathway

doi: 10.1538/expanim.24-0020

Figure Lengend Snippet: Ectodysplasin-A2 receptor (EDA2R) knockdown inhibits the activation of the NF-κB signaling pathway. (A) The levels of IκBα, p-IκBα (Ser32), NF-κB p65 and p-NF-κB p65 (Ser536) were detected in cardiomyocytes by western blotting. (B) The expression distribution of NF-κB p65 in cardiomyocytes was determined by immunofluorescence, the fluorescence intensity of p65 in the nucleus was quantified in three fields of three sections (the arrows represented the distribution of p65 in the nucleus, scale bar=50 µ m). (C) The transcriptional activity of NF-κB in cardiomyocytes was measured by the electrophoretic mobility shift assay (EMSA). **** P <0.0001 vs. con. #### P <0.0001. n=3.

Article Snippet: AC16 human cardiomyocytes were purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Knockdown, Activation Assay, Western Blot, Expressing, Immunofluorescence, Fluorescence, Activity Assay, Electrophoretic Mobility Shift Assay

Journal: Aging (Albany NY)

Article Title: Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology

doi: 10.18632/aging.103465

Figure Lengend Snippet: Differentially expressed genes in human cardiomyocytes. Numbers of differentially expressed genes after cyclic stretching: ( A ) Genes showing an at least 2-fold difference in expression; ( B ) genes showing an at least 1.5-fold difference in expression. Venn diagrams show the overlapping genes with at least 2-fold and 1.5-fold differences in expression.

Article Snippet: Human ventricular cardiomyocyte cells (AC16) were maintained in DMEM/F12 (GeneDireX Incorporation, Taiwan) supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, USA), 100 IU/mL penicillin (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology

doi: 10.18632/aging.103465

Figure Lengend Snippet: Functional analysis of differentially expressed genes in human cardiomyocytes after 1, 4, 12, 24, and 48 hours of cyclic stretching.

Article Snippet: Human ventricular cardiomyocyte cells (AC16) were maintained in DMEM/F12 (GeneDireX Incorporation, Taiwan) supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, USA), 100 IU/mL penicillin (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Functional Assay

Journal: Aging (Albany NY)

Article Title: Systems biology approach to exploring the effect of cyclic stretching on cardiac cell physiology

doi: 10.18632/aging.103465

Figure Lengend Snippet: Differential expression profile of 29 genes after 1, 4, 12, 24, and 48 hours of cyclical stretching in human cardiomyocytes. The heat map diagram shows expression changes for 29 genes that were altered in cardiac myocytes in response to at least three time points of mechanical stretching.

Article Snippet: Human ventricular cardiomyocyte cells (AC16) were maintained in DMEM/F12 (GeneDireX Incorporation, Taiwan) supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA, USA), 100 IU/mL penicillin (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma-Aldrich).

Techniques: Quantitative Proteomics, Expressing