Journal: Experimental Animals

Article Title: Transferrin receptor knockdown attenuates atrial fibrillation by inhibiting cardiomyocyte ferroptosis and atrial fibrosis

doi: 10.1538/expanim.24-0127

Figure Lengend Snippet: Transferrin receptor (TFRC) interference alleviates Angiotensin II-induced mouse HL-1 cell damage. (a) The interference efficiency of siRNA was detected by Real-time PCR and Western blot assay in mouse cardiomyocytes (HL-1 cells). (b) Relative mRNA and protein expression of TFRC in HL-1 cells. Data are shown as individual values with the error bar of SD. P -value: **<0.01. (c) Cell Counting Kit-8 assay was performed to determine the viability of HL-1 cells. (d) TFRC mRNA and protein expression in HL-1 cells were detected using Real-time PCR and Western blot, respectively. (e) The lipid reactive oxygen species (ROS) contents were detected using a C11-BODIPY581/591 fluorescent probe on flow cytometry. (f) Glutathione (GSH) levels and glutathione peroxidase (GSH-PX) activity. (g) acyl-CoA synthetase long chain family member 4 (ACSL4), Glutathione Peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) relative protein expression assayed by Western blot. (h) Real-time PCR measurement of Col1a2 and Col3a1 . Data are shown as individual values with the error bar of SD. P -value: **<0.01 vs. Control. ## <0.01 vs. Ang II + siNC. Ang II, Angiotensin II.

Article Snippet: Mouse cardiomyocytes (HL-1 cells) were obtained from iCell.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Cell Counting, Flow Cytometry, Activity Assay, Control

Journal: PLoS Computational Biology

Article Title: Tox_(R)CNN: Deep learning-based nuclei profiling tool for drug toxicity screening

doi: 10.1371/journal.pcbi.1006238

Figure Lengend Snippet: HL1 cells treated or not (-) with the indicated concentrations of compounds (μM) or DMSO were processed as described in the Materials and Methods. (A) Representative fluorescence microscopy images of DAPI-stained cells treated or not (Ctrl) with the highest concentrations of the indicated compounds in a reference experiment (Experiment #1) used for CNN training. (B-D) Boxplots of per-well toxicity assessments from established measurements: nucleus count (Num Nuc) (B), Caspase 3/7 nucleus:cytoplasm ratio (Casp nuc/cyto) (C), and Mitotracker cytoplasmic intensity (Mito) (D). (E) CNN architecture for predicting health status from single-cell image crops, as described in Materials and Methods. (F) Cropping strategies; representative image crops are shown of nucleus (Nuc), nucleus+cytoplasm (Cell), nucleus+margin (Nuc_Ring), and nucleus+cytoplasm+background+neighboring cells (All). (G) Boxplot of per-well toxicity assessment from CNN Nuc predictions (percentage of cells classified as healthy). (H) Plot displaying mean toxicity readouts of replicate wells, obtained from the percentage of healthy cells predicted by the different CNN models (CNN Nuc, Nuc_Ring, Cell, All, 4crops) and the standard nuclei counting (Num Nuc) for the different treatments indicated. For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. Points and corresponding error bars represent the mean and standard error of the mean, respectively, of results obtained by evaluating the 5 different CNN models trained for each cropping strategy. (I) Evaluation performance of the different CNN models for predicting toxic effects of staurosporine assessed using Caspase 3/7 fluorescent reporter as reference, as described in the Materials and Methods. Boxplots display AUC values obtained with the 5 models trained for each cropping strategy. (J) Correlations between cell density and CNN predictions obtained with the different models for untreated cells. Boxplots represent Pearson correlation coefficient, R, obtained with all 5 models trained with each cropping strategy (top), and exemplary dotplots for each strategy (selecting the model with the median AUC among the five) including regression line, R value and significance (bottom). p-value: *<0.01, **<0.001, ***<0.0001.

Article Snippet: Mouse cardiac muscle HL1 cells purchased from Merck Millipore were grown on fibronectin(25μg/ml)/gelatin(1mg/ml) coated dishes with 10% fetal bobine serum (FBS) in Claycomb medium (Sigma-Aldrich).

Techniques: Fluorescence, Microscopy, Staining

Journal: PLoS Computational Biology

Article Title: Tox_(R)CNN: Deep learning-based nuclei profiling tool for drug toxicity screening

doi: 10.1371/journal.pcbi.1006238

Figure Lengend Snippet: HL1 cells treated or not (-) with DMSO or the indicated concentrations of drugs (μM) from Experiment #1 were processed as described in the Materials and Methods. Plots display individual well toxicity readouts (top) and the 5-Fluorouracil dose-response curve fitted from well-averages, including the EC50 (bottom). Representative model results out of the 5 independent ones trained for each cropping strategy is shown. (A-C) CNN-based toxicity readouts: CNN Nuc (A), CNN Cell (B), and CNN All (C). (D-F) Standard toxicity readouts: Nuclei counting by standard image segmentation (Num Nuc) (D), mean Caspase 3/7 nucleus:cytoplasm ratio (Casp Nuc/Cyto) (E), and mean Mitotracker cytoplasmic intensity (Mito) (F). For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. Z-scores > 3 represent toxic hits.

Article Snippet: Mouse cardiac muscle HL1 cells purchased from Merck Millipore were grown on fibronectin(25μg/ml)/gelatin(1mg/ml) coated dishes with 10% fetal bobine serum (FBS) in Claycomb medium (Sigma-Aldrich).

Techniques:

Journal: PLoS Computational Biology

Article Title: Tox_(R)CNN: Deep learning-based nuclei profiling tool for drug toxicity screening

doi: 10.1371/journal.pcbi.1006238

Figure Lengend Snippet: (A) RCNN architecture for automated detection of cells and prediction of their health status from micrographs of DAPI fluorescence, as described in the Materials and Methods. (B) Example of nuclei bounding boxes resulting from the region proposal network included in the RCNN framework. (C) HL1 cells (Experiment #8) were seeded at the indicated densities (cells/well). (D,E) HL1 (Experiment #1) and EAHY926 (Experiment #9) cells were seeded at 5000 cells/well. (C-E) 24h after seeding, cells were treated or not (-) with the indicated concentrations of DMSO (%) or the indicated drugs (μM) and processed as described in the Materials and Methods. Representative images are shown of untreated cells at the indicated cell-seeding densities (cells/well). Plots display mean toxicity readouts of four replicate wells, obtained from the percentage of healthy cells predicted by the CNN Nuc (Tox_CNN) and RCNN (Tox_RCNN_balanced and Tox_RCNN) mixed models, and from nuclei counting by standard image segmentation (Num Nuc), or by using RCNN-based automated detection (Num Nuc RCNN) from Tox_RCNN training. For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values.

Article Snippet: Mouse cardiac muscle HL1 cells purchased from Merck Millipore were grown on fibronectin(25μg/ml)/gelatin(1mg/ml) coated dishes with 10% fetal bobine serum (FBS) in Claycomb medium (Sigma-Aldrich).

Techniques: Fluorescence

Journal: PLoS Computational Biology

Article Title: Tox_(R)CNN: Deep learning-based nuclei profiling tool for drug toxicity screening

doi: 10.1371/journal.pcbi.1006238

Figure Lengend Snippet: HL1 cells treated with one of 24 compounds or DMSO at the concentrations indicated (μM) (Experiments #11–14) were processed as described in the Materials and Methods. (A) Plots displaying mean toxicity readouts of four replicate wells, obtained from percentage of healthy cells predicted by the CNN Nuc (Tox_CNN) or RCNN (Tox_RCNN) mixed models, and from nuclei counting by standard image segmentation (Num Nuc), or by using RCNN-based automated detection (Num Nuc RCNN). For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. (B) Hierarchical clustering of features obtained with the Tox_CNN model from HL1 cells treated with 25μM of the indicated drugs or 0.78μM Taxol; untreated (-); DMSO, control. Colors highlight mechanism of toxicity associated with compounds.

Article Snippet: Mouse cardiac muscle HL1 cells purchased from Merck Millipore were grown on fibronectin(25μg/ml)/gelatin(1mg/ml) coated dishes with 10% fetal bobine serum (FBS) in Claycomb medium (Sigma-Aldrich).

Techniques: Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: New Molecular Mechanism Underlying Myc‐Mediated Cytochrome P450 2E1 Upregulation in Apoptosis and Energy Metabolism in the Myocardium

doi: 10.1161/JAHA.118.009871

Figure Lengend Snippet: Upregulation of cytochrome P450 2E1 (CYP 2E1) in multiple heart diseases and Myc activated CYP 2E1 gene promoter. CYP 2E1 expression ( A ) in heart tissues from patients with hypertrophic cardiomyopathy (HCM) (n=14) and normal controls (n=8), and quantitative analysis ( B ) using GAPDH for normalization (*** P <0.001 vs normal). CYP 2E1 expression ( C and D ) in heart tissues of the left anterior descending (LAD) coronary artery–, thoracic aorta constriction (TAC)–, and adriamycin‐induced mouse models, and quantitative analysis using GAPDH for normalization (* P <0.05 or ** P <0.01 vs sham or saline). CYP 2E1 expression ( E and F ) in HL ‐1 cells under starvation or isoprenaline stress, and quantitative analysis using GAPDH for normalization (** P <0.01 vs normal or saline). CYP 2E1 activity in vivo models ( G , * P <0.05 or ** P <0.01 vs sham or saline) and in vitro models ( H , * P <0.05 or ** P <0.01 vs normal or saline). Transcriptional activity of a series of truncated CYP 2E1 promoter constructs that transfected to HL ‐1 cells ( I , n=4 independent experiments; see Table for polymerase chain reaction primer sequences). Promoter activities of the p0.9cyp2e1‐luc and pGL 3‐Basic construct alone or cotransfected with the indicated amounts of Myc expression plasmid with or without Myc inhibitor 10058‐F4 ( J , n=3 independent experiments). Chromatin immunoprecipitation assay on extracts from HL ‐1 cells transfected with the Myc expression plasmid or with pc DNA 3 plamid as a control ( K , n=4 independent experiments). Transcriptional activity of a series of mutated CYP 2E1 promoter constructs that transfected to HL ‐1 cells ( L , n=4 independent experiments).

Article Snippet: Cells of HL‐1 adult mouse cardiac muscle were grown in Claycomb Medium supplemented with 10% FCS, 4 mmol/L l ‐glutamine, and 50 μmol/L noradrenaline (MedChem Express, HY‐13715B), in a humidified 5% CO 2 incubator as previously described.

Techniques: Expressing, Saline, Activity Assay, In Vivo, In Vitro, Construct, Transfection, Polymerase Chain Reaction, Plasmid Preparation, Chromatin Immunoprecipitation, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: New Molecular Mechanism Underlying Myc‐Mediated Cytochrome P450 2E1 Upregulation in Apoptosis and Energy Metabolism in the Myocardium

doi: 10.1161/JAHA.118.009871

Figure Lengend Snippet: Activation of the extracellular signal–regulated kinase (ERK) 1/2 and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway is pivotal for the expression of cytochrome P450 2E1 (CYP2E1) and Myc in HL ‐1 cells under stress. Expression of CYP 2E1, Myc, phosphorylated, and total level of ERK1/2, PI 3K, and Akt in heart tissues of thoracic aorta constriction (TAC)– and adriamycin‐induced mouse models ( A ). Quantitative analysis of protein expression using GAPDH for normalization ( B through F , ** P <0.01 or *** P <0.001 TAC vs sham; # P <0.05 or ## P <0.01 adriamycin vs saline). Expression of CYP 2E1, Myc, phosphorylated, and total level of ERK1/2 in HL ‐1 cells under starvation with or without mitogen‐activated protein kinase kinase/ ERK inhibitor ( G ). Quantitative analysis of protein expression using GAPDH for normalization ( H , ** P <0.01 or *** P <0.001 starvation vs PBS ; # P <0.05 or ## P <0.01 starvation+ PD 098059 vs starvation). Expression of CYP 2E1, Myc, phosphorylated, and total level of PI 3K and Akt in HL ‐1 cells under starvation with or without a PI 3K inhibitor ( I ). Quantitative analysis of protein expression using GAPDH for normalization ( J , ** P <0.01 or *** P <0.001 starvation vs PBS ; # P <0.05 or ## P <0.01 starvation+ LY 294002 vs starvation).

Article Snippet: Cells of HL‐1 adult mouse cardiac muscle were grown in Claycomb Medium supplemented with 10% FCS, 4 mmol/L l ‐glutamine, and 50 μmol/L noradrenaline (MedChem Express, HY‐13715B), in a humidified 5% CO 2 incubator as previously described.

Techniques: Activation Assay, Expressing, Saline

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: New Molecular Mechanism Underlying Myc‐Mediated Cytochrome P450 2E1 Upregulation in Apoptosis and Energy Metabolism in the Myocardium

doi: 10.1161/JAHA.118.009871

Figure Lengend Snippet: Inhibiting Myc blunted cytochrome P450 2E1 ( CYP 2E1) and consequently reduced Oxidative stress and apoptosis. Level of H 2 O 2 ( A ), malondialdehyde ( B ), glutathione ( C ), and total antioxidant capacity (T‐ AOC ) ( D ) in HL ‐1 cells under starvation with or without Myc inhibitor 10058‐F4 (* P <0.05 starvation vs PBS or starvation+100058‐F4 vs starvation). Mitochondrial cytochrome c (Cyt‐ c ) release and the activation of caspase 9 and caspase 3 were measured by Western blot in lysate from HL ‐1 cells under starvation with or without Myc inhibitor 10058‐F4 ( E ). Quantitative analysis of Cyt‐ c in cytoplasm and mitochondria ( F ). Quantitative analysis of activation of caspase 9 and caspase 3 using GAPDH for normalization ( G through J , * P <0.05 or ** P <0.01 starvation vs PBS or starvation+100058‐F4 vs starvation).

Article Snippet: Cells of HL‐1 adult mouse cardiac muscle were grown in Claycomb Medium supplemented with 10% FCS, 4 mmol/L l ‐glutamine, and 50 μmol/L noradrenaline (MedChem Express, HY‐13715B), in a humidified 5% CO 2 incubator as previously described.

Techniques: Activation Assay, Western Blot