human cardiomyocyte complete media with serum  (Celprogen Inc)

Journal: Nature Communications

Article Title: Reverse engineering of BNIP3 identifies a mitochondrial protective peptide

doi: 10.1038/s41467-026-73993-2

Figure Lengend Snippet: a Close proximity of BNIP3 to BAX or BAK1 in mouse embryonic fibroblasts (WT MEFs) (top) and in Bak knockout ( Bak −/− ) or Bax knockout ( Bax −/− ) MEFs (bottom). Interactions of BNIP3 and BAX or BAK1 are indicated by red dots with white arrows. Antibodies against BAX (2D2), BAK1 (4C2) and BNIP3 (1C8) were used along with a pair of secondary antibodies conjugated to complementary oligonucleotides in an in situ proximity ligation assay (PLA). (Nuclei stained with DAPI, blue; scale bars 30 µm and 10 µm, n = 3 independent experiments). b Close proximity of BNIP3 and BAX in isolated male mouse Bnip3 +/+ cardiomyocytes (mCM) (left) and in CM of male Bnip3-3xFlag-in ( Flag-Bnip3 ki/ki ) mice (right). ( n = 3 independent experiments). c Fluorescence-labelled recombinant mouse (m)BNIP3 incubated with a library of 13 synthesised BAX peptides immobilised on microarrays to reveal potential interaction sites in BAX. The colour coding ranges from white (low or no intensity), through yellow (medium intensity), to red (high intensity) (top). Structure of mBAX (PDB 4S0O) in ribbon representation with BNIP3 interaction sites coloured blue (α5), green (α6), yellow (α7) and orange (α8) (bottom). ( n = 3 identical subarrays with mouse IgG controls). d Modeller algorithm was used to predict the BNIP3 homology model based on BAX (PDB code 2K7W) as a template, followed by energy minimisation using NAMD 2.9 and CHARMM36 force field. The resulting model agreed well with the circular dichroism (top) and intramolecular cross-linking data obtained with recombinant human BNIP3 (hBNIP3). The DSSO cross-links identified in recombinant hBNIP3 by mass spectrometry were mapped onto the homology model (orange lines), supporting the predicted compact three-dimensional structure (bottom). e HDOCK docking of the hBNIP3 homology model and hBAX (PDB code 2k7w, model 1), with a docking score of −253.86 and a confidence score of 0.8887 (hit 1 of the top 10 optimal conformations). The predicted intermolecular hydrophobic interactions were mapped onto the docking model (magenta lines) (left). Furthermore, two hydrogen bonds were predicted (3D detail map) (right). Source data are provided as a Source Data file.

Article Snippet: To analyse the mitochondrial inner membrane potential in human iPSC-derived cardiomyocytes (Axol 2505), the cells were incubated in HEPES buffer (113 mM NaCl, 4.7 mM KCl, 12 mM HEPES, 1.2 mM MgSO 4 , 30 mM taurine, and 1.3 mM CaCl 2 , pH 7.4) under 1% O 2 at 37 °C for 2 h. For reoxygenation, the cells were incubated in HEPES buffer supplemented with 5.5 mM glucose under 21% O 2 at 37 °C for 2 h. During hypoxia/reoxygenation the cardiomyocytes were treated with B-017 (4.4 μM) or the negative control peptide (4.4 μM).

Techniques: Knock-Out, In Situ, Proximity Ligation Assay, Staining, Isolation, Fluorescence, Recombinant, Incubation, Circular Dichroism, Mass Spectrometry

Journal: Nature Communications

Article Title: Reverse engineering of BNIP3 identifies a mitochondrial protective peptide

doi: 10.1038/s41467-026-73993-2

Figure Lengend Snippet: a Cellular response to B-017 treatment in different cell lines exposed to cell death signalling stimulation using live cell impedance measurements. HEK293 cells (8000 cells per well) and human cardiac fibroblasts (HCF; 2000 cells per well) were treated simultaneously with 100 nM or 10 nM staurosporine (STS) and B-017 (52 µM) or vehicle (Veh.) after reaching a cell index of 1, indicating a stable adhesion to a microtiter plate consisting of gold microelectrodes fused to the bottom of the surface. The time point chosen to assess the differences was 1 h after treatment for HEK293 cells and 4 h for HCF to exclude a proliferation effect. ( n = 3 independent experiments). b – f Cellular consequences of B-017 treatment in MCF-7 cells, mouse cardiac fibroblasts (mCF), human cardiomyocytes (HCM), HEK293 cells, and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), related to b caspase 3/7 activity (MCF-7: n = 3 independent experiments; mCF: n = 5 independent experiments), c reactive oxygen species generation (HCM: n = 6 independent experiments; HEK293: n = 4 independent experiments), d mitochondrial Ca 2+ overload ( n = 5 independent experiments), e formation of the mitochondrial permeability transition pore (mPTP) ( n = 6 independent experiments), and f mitochondrial membrane depolarisation (ΔΨ mito ) (HCM: n = 6 independent experiments and iPSC-CM: n = 5 (Veh., Veh. in H/R) and n = 3 (B-017 in H/R) independent experiments. STS (500 nM, 2 µM), doxorubicin (DOX, 10 µM), and hypoxia/reoxygenation (H/R) were chosen as cell death signalling inducing agents; B-017 (52 µM) or vehicle was administered concomitantly with the stimulus. Luminescence intensity of caspase-Glo 3/7 reagent was assessed using a luminescence microplate reader, and fluorescence intensity of H 2 DCFDA (H 2 O 2 ), rhodamine-2 AM (Ca 2+ mito ), TMRM (ΔΨ mito ), and calcein (mPTP) was measured using a fluorescence microplate reader. Fluorescence intensity of H 2 DCFDA (H 2 O 2 ) and JC-1 (ΔΨ mito ) was determined by fluorescence microscopy. One-way ANOVA, data are presented as mean ± SD. Source data are provided as a Source Data file.

Article Snippet: To analyse the mitochondrial inner membrane potential in human iPSC-derived cardiomyocytes (Axol 2505), the cells were incubated in HEPES buffer (113 mM NaCl, 4.7 mM KCl, 12 mM HEPES, 1.2 mM MgSO 4 , 30 mM taurine, and 1.3 mM CaCl 2 , pH 7.4) under 1% O 2 at 37 °C for 2 h. For reoxygenation, the cells were incubated in HEPES buffer supplemented with 5.5 mM glucose under 21% O 2 at 37 °C for 2 h. During hypoxia/reoxygenation the cardiomyocytes were treated with B-017 (4.4 μM) or the negative control peptide (4.4 μM).

Techniques: Derivative Assay, Activity Assay, Permeability, Membrane, Fluorescence, Microscopy

Journal: Frontiers in Toxicology

Article Title: Evaluation and investigation of the cardiotoxicity of the potential anti-cholangiocarcinoma drug lanatoside C

doi: 10.3389/ftox.2026.1783444

Figure Lengend Snippet: Impact of Lan C on cell viability of AC16 cells evaluated by CCK-8 assay. (A) Cell viability of AC16 cells treated with the indicated concentrations of Lan C for 12, 24, and 48 h. (B) Dose-response curve of cell inhibition on AC16 cell proliferation after 48-h treatment. The half maximal inhibitory concentration (IC50) is presented. **** P < 0.0001.

Article Snippet: Human AC16 cardiomyocytes (ATCC® CRL-3568TM) were employed for in vitro studies, with untreated cells included as negative controls.

Techniques: CCK-8 Assay, Inhibition, Concentration Assay

Journal: Frontiers in Toxicology

Article Title: Evaluation and investigation of the cardiotoxicity of the potential anti-cholangiocarcinoma drug lanatoside C

doi: 10.3389/ftox.2026.1783444

Figure Lengend Snippet: Effects of Lan C on Cell Viability and Reactive Oxygen Species (ROS) Levels in AC16 Cells. (A) Representative confocal microscopy images showing ROS-associated fluorescence intensity in cells treated with the indicated concentrations of Lan C for 48 h. (B) ROS fluorescence intensity was measured by flow cytometry in cells treated with the indicated concentrations of Lan C for 48 h. (C) Quantitative analysis of ROS fluorescence intensity obtained from flow cytometry. Data are presented as mean ± SD. ** P < 0.005, **** P < 0.0001. Scale bars: 100 μm.

Article Snippet: Human AC16 cardiomyocytes (ATCC® CRL-3568TM) were employed for in vitro studies, with untreated cells included as negative controls.

Techniques: Confocal Microscopy, Fluorescence, Flow Cytometry

Journal: Frontiers in Toxicology

Article Title: Evaluation and investigation of the cardiotoxicity of the potential anti-cholangiocarcinoma drug lanatoside C

doi: 10.3389/ftox.2026.1783444

Figure Lengend Snippet: Analysis of mitochondrial-mediated apoptotic pathway protein expression and lactate dehydrogenase (LDH) release in AC16 cells following Lan C treatment. (A) Representative Western blot bands showing protein expression levels of Bcl-2, cytochrome c, cleaved caspase-9, and cleaved caspase-3. (B–E) Quantitative analysis of the relative band intensity (gray value) for Bcl-2 (B) , cytochrome c (C) , cleaved caspase-9 (D) , and cleaved caspase-3 (E) . (F) The release rate of LDH into the extracellular medium after AC16 cells were treated with Lan C. * P < 0.05; ** P < 0.005; **** P < 0.0001.

Article Snippet: Human AC16 cardiomyocytes (ATCC® CRL-3568TM) were employed for in vitro studies, with untreated cells included as negative controls.

Techniques: Expressing, Western Blot