h9c2 immortalized rat cardiac myoblasts  (ATCC)

Journal: bioRxiv

Article Title: Cells Engage Endogenous Malonate Synthesis to Drive Mitochondrial Metabolism

doi: 10.64898/2026.05.22.727248

Figure Lengend Snippet: (A-B) Whole cell lysates or mitochondrial enriched lysates (MCAT, CS blots) from (A) C2C12 mouse myoblast or (B) H9c2 rat cardiac myoblast clonal cell lines of the indicated genotypes expressing mtDSRed or Mcat were immunoblotted for the indicated protein targets. Data are representative of 3 biological replicates. Ctrl = control, CS = citrate synthase, DLAT = dihydrolipoamide s-acetyltransferase, DLST = dihydrolipoamide S-succinyltransferase, SDHB = succinate dehydrogenase B, ATP5a = mitochondrial loading control, β actin = whole cell loading control ( C-D ) Blue-Native PAGE of crude isolated mitochondrial lysates from (C) C2C12 and (D) H9c2 clonal cell lines of the indicated genotypes expressing mtDSRed or Mcat, immunoblotted for the indicated ETC complex subunits. Data are representative of 3 biological replicates. ( E ) Diagram depicting peptide products produced by digest of NDUFAB1 with AspN endoproteinase and resulting ions detected by mass spectrometry. ( F ) Relative abundance of octanoyl-NDUFAB1 normalized to holo-NDUFAB1 in the indicated cell lines. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post-hoc test, * = p<0.05 (n=3). ( G-H ) Mass isotopologue distribution of octanoyl-NDUFAB1 in (G) wild-type C2C12 cells or (H) wild-type H9c2 cells after culture with uniformly labeled U- 13 C 3 malonate for 3 hours (n=3). ( I-J ) Mass isotopologue distribution of holo-NDUFAB1 in (I) wild-type C2C12 cells or (J) wild-type H9c2 cells after culture with uniformly labeled malonate for 3 hours (n=3). Error bars represent +/- 1 SEM.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC) and H9c2 immortalized rat cardiac myoblasts (ATCC CRL-1446, verification provided by ATCC) were grown in DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) and 10% FBS (Sigma, F0926) at 37°C, 5% CO 2 .

Techniques: Expressing, Control, Blue Native PAGE, Isolation, Produced, Mass Spectrometry, Labeling

Journal: bioRxiv

Article Title: Cells Engage Endogenous Malonate Synthesis to Drive Mitochondrial Metabolism

doi: 10.64898/2026.05.22.727248

Figure Lengend Snippet: (A) Whole cell lysates or mitochondrial enriched lysates (MCAT, CS) in clonal H9c2 cell lines of the indicated genotypes. (B) Blue-native PAGE of crude isolated mitochondrial lysates from the indicated H9c2 cell lines expressing mtDSRed or Mcat, immunoblotted for the indicated ETC complex subunits. Data are representative of 3 biological replicates. (C-E) Whole cell lysates from (C) wild-type C2C12 cells or (D) wild-type H9c2 cells or (E) C2C12 cells of the indicated genotype expressing mtDSRed or Mcat, expressing GFP or NDUFAB1-FLAG(ires)GFP blotted with the indicated antibodies. (F) Extracted ion chromatograms of ions corresponding to holo-NDUFAB1 or octanoyl-NDUFAB1 from the indicated sample and HCD collision energy (%). (G) Table of quantified ions for the indicated NDUFAB1 modification after AspN digest and the corresponding approach by mass spectrometry for detection in a 15-minute C18 reversed phase liquid chromatography method. (H) Mass isotopologue distribution of precursor ion for holo-NDUFAB1 detected in control cell line (n=3) after culture in uniformly labeled 13 C malonate for 3 hours. (I) Mass isotopologue distribution of precursor ion for octanoyl-NDUFAB1 detected in a control cell line (n=3) after culture in uniformly labeled 13 C malonate for 3 hours, corrected with unlabeled sample. Error bars represent +/- 1 SEM.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC) and H9c2 immortalized rat cardiac myoblasts (ATCC CRL-1446, verification provided by ATCC) were grown in DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) and 10% FBS (Sigma, F0926) at 37°C, 5% CO 2 .

Techniques: Blue Native PAGE, Isolation, Expressing, Modification, Mass Spectrometry, Reversed-phase Chromatography, Control, Labeling

Journal: bioRxiv

Article Title: Cells Engage Endogenous Malonate Synthesis to Drive Mitochondrial Metabolism

doi: 10.64898/2026.05.22.727248

Figure Lengend Snippet: ( A ) Whole cell lysates from the indicated cell lines, separated by SDS-PAGE and immunoblotted with the indicated antibodies. ( B ) Relative abundance of octanoyl-NDUFAB1 normalized to holo-NDUFAB1 in the indicated clonal C2C12 cell lines. Statistical analysis performed by one-way ANOVA followed by Tukey’s post-hoc test, (n=3). ns = p>0.05. ( C ) Ratio of octanoyl-NDUFAB1 to holo-NDUFAB1 expressed as a percent of the matched control in the indicated cell line (n=3). ( D ) Mass isotopologue distribution of octanoyl-NDUFAB1 relative to holo-NDUFAB1 in the indicated cell line after culture with uniformly labeled malonate for 24 hours (n=3) ( E ) Overlapping mass isotopologue distribution of even isotopologues from the indicated cell lines. Statistical analysis done by two-way ANOVA with Dunnet’s multiple comparisons test (n=3). ns = p>0.05, * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. ( F ) Mass isotopologue distribution of octanoyl-NDUFAB1 in wild-type C2C12 cells after culture with uniformly labeled malonate for the indicated period of time (n=3), expressed as a percent of total pool (%). ( G ) Mass isotopologue distribution of octanoyl-NDUFAB1 in wild-type H9c2 cells after culture with uniformly labeled malonate for the indicated period of time (n=3), expressed as a percent of total pool (%). Error bars represent +/- 1 SEM.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC) and H9c2 immortalized rat cardiac myoblasts (ATCC CRL-1446, verification provided by ATCC) were grown in DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) and 10% FBS (Sigma, F0926) at 37°C, 5% CO 2 .

Techniques: SDS Page, Control, Labeling

Journal: bioRxiv

Article Title: Cells Engage Endogenous Malonate Synthesis to Drive Mitochondrial Metabolism

doi: 10.64898/2026.05.22.727248

Figure Lengend Snippet: ( A) Mass isotopologue distribution of octanoyl-NDUFAB1 in wild-type H9c2 cells after culture with uniformly labeled glucose for 48 hours in the presence of 1 mM malonate (n=3), expressed as a percent of total pool (%). (B) Mass isotopologue distribution of octanoyl-NDUFAB1 in wild-type H9c2 cells after culture with uniformly labeled glucose for 48 hours without additional malonate (n=3), expressed as a percent of total pool (%). (C) Fractional enrichment (%) of all isotopologues of holo-NDUFAB1 in cells of the indicated lineage grown with or without 1 mM malonate and labeled with [U- 13 C 6 ]-glucose for 48 hours (n=3). (D) Mass isotopologue distribution of octanoyl-NDUFAB1 in wild-type H9c2 cells after culture with uniformly labeled glucose for 24 hours in the presence of 15 µM malonate (n=3), expressed as a percent of total pool (%). (E) Mass isotopologue distribution of octanoyl-NDUFAB1 in wild-type H9c2 cells after culture with 15 µM uniformly labeled malonate for 24 hours (n=3), expressed as a percent of total pool (%). Error bars represent +/- 1 SEM.

Article Snippet: C2C12 immortalized mouse skeletal myoblasts (ATCC CRL-1772, verification provided by ATCC) and H9c2 immortalized rat cardiac myoblasts (ATCC CRL-1446, verification provided by ATCC) were grown in DMEM with 4.5 g/L glucose, 4 mM glutamine, and sodium pyruvate (Corning, 10-013-CV) and 10% FBS (Sigma, F0926) at 37°C, 5% CO 2 .

Techniques: Labeling

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Metformin alleviates diastolic dysfunction in mice with experimental diabetic cardiomyopathy

doi: 10.1016/j.jmccpl.2025.100825

Figure Lengend Snippet: Metformin treatment does not attenuate inflammation in mice with T2D or H9C2 cardiac myoblasts. (A) Relative mRNA expression of pro-inflammatory cytokines ( Il6 , Il1b and Tgfb ) and chemokines ( Ccl2 and Ccl5 ) normalized to Ppia mRNA expression in myocardial extracts of lean mice and mice with T2D following a 7.5-wk treatment with vehicle control (VC) or metformin (n = average of 3 technical replicates from 5 to 11 mice/group). (B) Representative immunofluorescence images of H9C2 cells stained with nuclear factor κ B (NFκB) and 4′,6-diamidino-2-phenylindole (DAPI) (C) Integrated density of NFκB (n = average of 10 myoblasts from 3 biological replicates), (D) percent nuclear area of NFκB expression (n = average of 10 myoblasts from 3 biological replicates), (E) cell viability determined via MTT assay (n = average of 6 technical replicates from 3 biological replicates/group), (F) representative immunofluorescence images of H9C2 cells stained with WGA and DAPI and (G) relative cardiomyocyte (CM) cross-sectional area (CSA) (n = average of 35–60 cardiac myoblasts from 3 biological replicates/group), following a 1-hr incubation with either vehicle (sterile PBS) or lipopolysaccharide (LPS; 1 μg/mL) with or without metformin (5 or 10 μM) (Scale bar, 50 μm). Values represent mean ± SEM. Differences were determined using a two-way ANOVA followed by a Bonferroni post-hoc analysis (A) or a one-way ANOVA followed by a Bonferroni post-hoc analysis (C–E, G). # P < 0.05, significantly different versus respective lean counterpart. ^ P < 0.05, significantly different versus VC-treated H9C2 cells. Ccl2, chemokine ligand 2; Ccl5, chemokine ligand 5; Il1b , interleukin 1b; Il6, interleukin 6; Ppia , peptidylprolyl isomerase A; Tgfb , transforming growth factor B.

Article Snippet: H9C2 rat cardiac myoblasts (ATCC CRL-1446) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco 2764658) supplemented with 10 % fetal bovine serum (v/v) (Gibco 16000044) and 1 % penicillin-streptomycin (v/v) (Gibco 15140122) at 37 °C in a humidified 5 % CO 2 (v/v) atmosphere.

Techniques: Expressing, Control, Immunofluorescence, Staining, MTT Assay, Incubation, Sterility

Journal: Physiological Reports

Article Title: Dietary influences on chemotherapy sensitivity and cardiotoxicity modulated by IRE1 targeting in triple‐negative breast cancer in female mice

doi: 10.14814/phy2.70400

Figure Lengend Snippet: IRE1 blockade modulates mitochondria respiration. (a) Hearts oxidative stress from control and Western diets‐fed mice were measured by 4HNE staining. Representative image of 4HNE staining hearts from control (b) and Western (c) fed mice n = 6–8, analyzed by two‐way ANOVA followed by a Tukey's multiple comparison test (Scale bar = 100 μm). H9C2 rat cardiomyoblasts were plated and treated them with 100 nM IRE1 morpholino for 24 h. Then, cells were treated with or without 0.25 μM DOX. (d) Cell viability was measured by fluorescent microscopy (BioTek Cytation, Agilent Technologies) or Cell Imaging Multi‐Mode Reader, combining fluorescence and high contrast brightfield imaging with conventional multi‐mode detection. (e–g) Role of IRE1 in mitochondria respiration. A respirometry experiment was performed to measure the OCR which is a measure of mitochondrial respiration on H9C2 cells. (e) OCR measure, (f) basal OCR, and (g) maximal OCR. OCR was normalized to cell number; n = 3; two‐way ANOVA followed by a Tukey's multiple comparison test. 4HNE, 4‐hydroxy 2‐nonenal; DOX, doxorubicin; IRE1M, inositol‐requiring enzyme‐1 antisense morpholino; OCR, oxygen consumption rate.

Article Snippet: H9C2 rat cardiac myoblast cell line was obtained from ATCC (CRL‐1446) and cultured in DMEM media supplemented with 10% FBS, penicillin/streptomycin, and glutamine, kept at 37°C and 5% CO 2 .

Techniques: Control, Western Blot, Staining, Comparison, Microscopy, Imaging, Fluorescence

Journal: ACS Omega

Article Title: Chemical Constituents from the Leaves of Eruca sativa (Rocket Salad) Exhibiting Dual Cardioprotective and Antiplatelet Activities

doi: 10.1021/acsomega.5c05585

Figure Lengend Snippet: Antioxidant and antiapoptotic effects of compound 2 in H9c2 cardiomyocytes under hypoxia/reoxygenation (H/R) conditions. (A) Intracellular ROS levels were assessed using CM-H 2 DCFDA staining followed by flow cytometry. H9c2 cells were pretreated with compound 2 (1 or 2 mM) for 24 h and then subjected to H/R (1 h hypoxia/4 h reoxygenation) stress. Representative histograms (top) and quantification of relative fluorescence intensity (RFI, bottom) are shown. (B) Apoptotic cell death was evaluated by Annexin V-FITC and propidium iodide (PI) double staining. H9c2 cells pretreated with compound 2 were exposed to H/R, and apoptotic populations were quantified by flow cytometry. Representative dot plots (top) and the percentage of apoptotic cells (bottom) are shown. Quantitative data are presented as mean ± standard deviation ( n = 4); *** p < 0.001.

Article Snippet: H9c2 rat cardio-myoblast cell (CRL-1446, ATCC, Manassas, VA, USA) was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% FBS and 1% A/A in a humidified 5% CO 2 atmosphere.

Techniques: Staining, Flow Cytometry, Fluorescence, Double Staining, Standard Deviation