transfection ac16 human cardiomyocytes  (Procell Inc)

Journal: Frontiers in Physiology

Article Title: MiR-200a-3p protects against myocardial ischemia-reperfusion injury via KEAP1–NRF2 signaling

doi: 10.3389/fphys.2026.1826306

Figure Lengend Snippet: Assessment of miR-200a-3p function in AC16 cardiomyocytes under hypoxia–reoxygenation (H/R). (A) miR-200a-3p expression was quantified by quantitative real-time PCR following transfection with miR-200a-3p mimic, inhibitor, or corresponding negative controls under normoxic or H/R conditions. (B) Cell viability was measured using the CCK-8 assay. (C, D) Apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. (E, F) Intracellular reactive oxygen species (ROS) levels were assessed using the DCFH-DA fluorescent probe with flow cytometric analysis. (G, H) Oxidative stress markers, including malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, were determined in cell lysates. (I, J) Secretion of inflammatory cytokines TNF-α and IL-1β into culture supernatants was measured by ELISA assay. Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *** p < 0.001, vs. Control, ### p < 0.001, vs. H/R + NC mimic, && p < 0.01, &&& p < 0.001, vs. H/R + NC inhibitor.

Article Snippet: Human AC16 cardiomyocyte−like cells (Cat. No. CRL−3568; ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, CCK-8 Assay, Flow Cytometry, Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Frontiers in Physiology

Article Title: MiR-200a-3p protects against myocardial ischemia-reperfusion injury via KEAP1–NRF2 signaling

doi: 10.3389/fphys.2026.1826306

Figure Lengend Snippet: Validation of KEAP1 as a direct target of miR-200a-3p. (A) Schematic representation of the predicted binding site of miR-200a-3p within the 3′-UTR of human KEAP1 mRNA, as identified by TargetScan. Both the wild-type (WT) and mutant (MUT) sequences used for dual-luciferase reporter assays are shown. (B) Dual-luciferase reporter assays were performed in HEK293T cells co-transfected with WT- or MUT-KEAP1 3′-UTR plasmids and either miR-200a-3p mimic or NC mimic. Firefly luciferase activity was normalized to Renilla luciferase activity, and relative luciferase activity was calculated. Statistical analyses were performed using Student’s t-test. *** p < 0.001, vs. NC mimic. (C, D) AC16 cells were transfected with miR-200a-3p mimic, inhibitor, or their corresponding controls and then subjected to H/R treatment. KEAP1 expression was assessed by quantitative real-time PCR (C) and western blotting (D) . Protein bands were quantified using ImageJ software, and GAPDH was used as the internal reference. All data are presented as mean ± SD from at least three independent experiments. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. *** p < 0.001, vs. control; ### p < 0.001, vs. H/R + NC mimic, && p < 0.01, &&& p < 0.001, vs. H/R + NC inhibitor.

Article Snippet: Human AC16 cardiomyocyte−like cells (Cat. No. CRL−3568; ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Biomarker Discovery, Binding Assay, Mutagenesis, Luciferase, Transfection, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software, Control

Journal: Frontiers in Physiology

Article Title: MiR-200a-3p protects against myocardial ischemia-reperfusion injury via KEAP1–NRF2 signaling

doi: 10.3389/fphys.2026.1826306

Figure Lengend Snippet: KEAP1 mediates the protective effects of miR-200a-3p against H/R-induced oxidative injury in AC16 cardiomyocytes. (A, B) Western blot analysis of KEAP1 protein expression in AC16 cardiomyocytes transfected with si-KEAP1 or KEAP1 overexpression plasmid under H/R conditions, confirming the efficiency of KEAP1 knockdown and overexpression. *** p < 0.001, vs. H/R + si-NC; ### p < 0.001, vs. H/R + Vector; (C) Cell viability assessed by CCK-8 assay in AC16 cardiomyocytes. (D) Flow cytometric analysis of apoptosis using Annexin V/PI staining in AC16 cardiomyocyte. (E) Intracellular ROS levels measured by flow cytometry in AC16 cardiomyocytes. (F, G) Oxidative stress–related biochemical indices, including MDA levels (F) and SOD activity (G) , in AC16 cardiomyocytes under the indicated experimental conditions. (H, I) ELISA analysis of pro-inflammatory cytokines TNF-α (H) and IL-1β (I) in culture supernatants of AC16 cardiomyocyte. Data are presented as mean ± SD from at least three independent experiments. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. *** p < 0.001, vs. control; ### p < 0.001, vs. H/R, & p < 0.05, && p < 0.01, &&& p < 0.001, vs. H/R + miR-200a-3p mimic.

Article Snippet: Human AC16 cardiomyocyte−like cells (Cat. No. CRL−3568; ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Knockdown, CCK-8 Assay, Staining, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Physiology

Article Title: MiR-200a-3p protects against myocardial ischemia-reperfusion injury via KEAP1–NRF2 signaling

doi: 10.3389/fphys.2026.1826306

Figure Lengend Snippet: MiR-200a-3p promotes Nrf2/ARE signaling by targeting KEAP1 in cardiomyocytes. (A, B) Immunofluorescence staining of Nrf2 in AC16 cardiomyocytes under the indicated conditions. Representative images show Nrf2 localization in the nucleus and cytoplasm following H/R injury with miR-200a-3p overexpression, KEAP1 silencing, or combined miR-200a-3p mimic and KEAP1 overexpression. Nuclei were counterstained with DAPI. (C–G) Western blot analysis of nuclear and cytoplasmic Nrf2, as well as the Nrf2 downstream antioxidant enzymes HO-1 and NQO1, in AC16 cardiomyocytes subjected to H/R and genetic modulation of miR-200a-3p and KEAP1. Data are presented as mean ± SD from at least three independent experiments. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. *** p < 0.001, vs. control; ### p < 0.001, vs. H/R, & p < 0.05, && p < 0.01, &&& p < 0.001, vs. H/R + miR-200a-3p mimic.

Article Snippet: Human AC16 cardiomyocyte−like cells (Cat. No. CRL−3568; ATCC, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO 2 .

Techniques: Immunofluorescence, Staining, Over Expression, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Limonin alleviates pyroptosis and inflammatory responses in cardiomyocytes of myocardial ischemia-reperfusion mice by inhibiting the caspase-3/GSDME pathway

doi: 10.3389/fimmu.2026.1704424

Figure Lengend Snippet: Limonin inhibits pyroptosis in cardiomyocyte AC16 cells. (A) CCK8 was used to detect the effects of limonin on the viability of AC16 cells after incubation with different concentrations of limonin for 24 h, and the OGD/R cell model was established after incubation (n = 5). (B) CCK8 was used to detect the effects of limonin (50 μmol/L) and triclabendazole (40 μmol/L) treatment for 24 h followed by OGD/R modeling on AC16 cells (n = 5). (C) PI/Hoechst 33258 double staining was used to detect the changes in pyroptosis (×100, Scale bar: 50 μm, n = 5). (D) Bar chart for statistical analysis of PI/Hoechst 33258. (E) Flow cytometry was used to detect the changes in pyroptosis (n = 5). ns represents no statistical significance compared with the control group, and * represents comparison between two groups (* P < 0.05, ** P < 0.01, *** P < 0.001*, **** P < 0.0001).

Article Snippet: Human cardiomyocytes AC16 cells were purchased from Wuhan Procell Life Science & Technology Co. Ltd. (Wuhan, China).

Techniques: Incubation, Double Staining, Flow Cytometry, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Limonin alleviates pyroptosis and inflammatory responses in cardiomyocytes of myocardial ischemia-reperfusion mice by inhibiting the caspase-3/GSDME pathway

doi: 10.3389/fimmu.2026.1704424

Figure Lengend Snippet: Limonin can improve tissue damage and inflammatory response in I/R mice. (A) TUNEL staining was used to detect the changes in myocardial tissue apoptosis (×100, Scale bar: 100 μm, n = 10). (B) HE staining was used to detect pathological changes in the myocardial tissue (×100, Scale bar: 100 μm, n = 10). (C) Masson staining was used to observe changes in the myocardial tissue (×100, Scale bar: 100 μm, n = 10). (D) ELISA was used to detect changes in inflammatory factors IL-6, IL-1β, TNF-α, and IL-10 in the supernatant of AC16 cells (n = 5). (E) ELISA was also used to detect changes in inflammatory factors IL-6, LI-1β, TNF-α, and IL-10 in mouse blood (n = 10). (F) Relative expression levels of IL-6, IL-1β, TNF-α, and IL-10 mRNA in mouse myocardial tissues detected by RT-qPCR. (G) Relative expression levels of IL-6, IL-1β, TNF-α, and IL-10 mRNA in AC16 cells detected by RT-qPCR. * represents comparison between two groups (* P < 0.05, ** P < 0.01, *** P < 0.001*, **** P < 0.0001).

Article Snippet: Human cardiomyocytes AC16 cells were purchased from Wuhan Procell Life Science & Technology Co. Ltd. (Wuhan, China).

Techniques: TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Comparison

Journal: Frontiers in Immunology

Article Title: Limonin alleviates pyroptosis and inflammatory responses in cardiomyocytes of myocardial ischemia-reperfusion mice by inhibiting the caspase-3/GSDME pathway

doi: 10.3389/fimmu.2026.1704424

Figure Lengend Snippet: Limonin can inhibit the Caspase-3/GSDME signaling pathway. (A) Relative expression levels of caspase-3, cleaved-caspase-3, GSDME-F, GSDME-N, and IL-18 proteins in myocardial tissues detected by western blot experiment (n = 5). (B) Relative expression levels of caspase-3, cleaved-caspase-3, GSDME-F, GSDME-N, and IL-18 proteins in AC16 cells detected by western blot experiment (n = 5). (C) Detection of GSDME-N expression levels in AC16 cells by immunofluorescence assay (200×, scale bar: 50 μm, n = 5) (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Human cardiomyocytes AC16 cells were purchased from Wuhan Procell Life Science & Technology Co. Ltd. (Wuhan, China).

Techniques: Expressing, Western Blot, Immunofluorescence