d10  (ZeptoMetrix corporation)

Journal: bioRxiv

Article Title: Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

doi: 10.64898/2026.01.11.698907

Figure Lengend Snippet: (A) Outline of cell isolation and FACS of SSC-A Hi /GFP Hi ventricular cardiomyocytes from adult male zebrafish. (B) Representative laser confocal z-stack images of a sorted cardiomyocyte showing the colocalization of two sarcomeric proteins. α-Actinin staining is shown in cyan hot, Titin in orange hot, and nuclear staining in grey. The image on the right shows a magnified (3x zoom) area. (C) Representative laser confocal z-stack images of a sorted cardiomyocyte showing the sarcomeric localization of α-Actinin and myomesin. α-Actinin staining is shown in cyan hot, Myomesin in orange hot, and nuclear staining in grey. The images on the right (c’ and c’’) show 3x magnified regions highlighting the M-bands and Z-discs. (D) Normalized fluorescence intensity profile showing sarcomeric α-Actinin and Myomesin staining along the depicted line shown in c’’. The M-bands are labeled by Myomesin and the Z-discs by α-Actinin.

Article Snippet: Conjugated and primary antibodies used in this study include anti-Cardiac Troponin T (cTnT) Mouse Monoclonal Antibody (13-11, 1:500; ThermoFisher Scientific); Alexa Fluor® 647 Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), PE Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), anti-α-Actinin (Sarcomeric) Vio® R667, REAfinityTM (1:200; Miltenyi Biotec), anti-TTN (Titin) monoclonal antibody (M07), clone 2F12 (mouse, 1:250, Abnova), anti-Myomesin (mouse, mMaC myomesin B4, 1:200, DSHB).

Techniques: Cell Isolation, Staining, Fluorescence, Labeling

Journal: bioRxiv

Article Title: Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

doi: 10.64898/2026.01.11.698907

Figure Lengend Snippet: (A) Outline of adult male zebrafish ventricle isolation, dissociation, cytospin, and subsequent laser confocal imaging. (B) Representative laser confocal images of GFP+ cardiomyocytes showing intact and high-quality cells with visible sarco-meres. GFP fluorescence is shown in cyan, while nuclei are shown in yellow. (C) Representative laser confocal images of GFP+ cardiomyocytes showing intact cells with variable morphological shapes and sizes. Four main morphological features are highlighted, including rounded, rod-shaped, multipolar, and elongated cardiomyocytes. (D) Outline of cell isolation and FACS of GFP+ ventricular cardiomyocytes from adult male zebrafish. (E) Representative laser confocal images of FACS-isolated cardiomyocytes showing α-Actinin and myomesin staining. α-Actinin staining is shown in cyan hot, myomesin in orange hot, and nuclear staining in grey. The image on the right shows a resulting mask from the staining image on the left using Fiji. (F) Masks of randomly selected cardiomyocytes from (E) showing marked differences in the morphology and shape of cardiomyocytes. (G) Quantitative comparisons of perimeter, MinFeret, Circularity, and Aspect Ratio descriptors are shown (each group representing N = 6), revealing heterogeneity in male cardiomyocyte size and shape. (H) Outline of adult male zebrafish ventricle isolation, dissociation, and live-cell acoustic flow cytometry and bright field imaging of the GFP Hi SSC-A Hi cardiomyocyte cluster. Cells were run without fixation. (I) Representative bright field images of GFP Hi SSC-A Hi cardiomyocytes obtained using Attune CytPix acoustic focus.

Article Snippet: Conjugated and primary antibodies used in this study include anti-Cardiac Troponin T (cTnT) Mouse Monoclonal Antibody (13-11, 1:500; ThermoFisher Scientific); Alexa Fluor® 647 Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), PE Mouse anti-Cardiac Troponin T (cTnT), (1:250; BD Biosciences), anti-α-Actinin (Sarcomeric) Vio® R667, REAfinityTM (1:200; Miltenyi Biotec), anti-TTN (Titin) monoclonal antibody (M07), clone 2F12 (mouse, 1:250, Abnova), anti-Myomesin (mouse, mMaC myomesin B4, 1:200, DSHB).

Techniques: Isolation, Imaging, Fluorescence, Cell Isolation, Staining, Flow Cytometry

Journal: Cell Death Discovery

Article Title: Inhibition of TBK1/IKKε mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing

doi: 10.1038/s41420-025-02841-x

Figure Lengend Snippet: A Phosphoproteome clustering of significantly regulated sites following TNF and T cell treatment of polyclonal pools of sgCtrl or sgTRAF2-transduced D10 cells. Values represent T0 normalized phosphorylation changes. The sample dendrogram is split vertically to highlight the outmost cluster of samples. All sample conditions are included in the heatmap. B Overlap of T cell and TNF response in sensitized tumor cells. Phosphorylation changes in sgTRAF2 D10 cells following 6 h of T cell co-culture vs. sgTRAF2 at T0. Red color coding indicates shared phosphorylation sites that are coordinately regulated in sgTRAF2 D10 cells following 4-h TNF treatment. C Enrichment analysis of phosphorylation sites from the most strongly induced phosphorylation cluster in ( A ). D t-SNE analysis of significantly regulated proteins and phosphorylation sites in T cell and TNF treatment time course described in ( A ). E Enrichment analysis of phosphorylation sites significantly upregulated in sgCtrl BLM cells treated with T cells + Birinipant but not upregulated in sgTRAF2 BLM cells treated under the same conditions. F Phosphorylation changes in TNFR1-associated proteins from BLM cells treated with T cells ± Birinipant (1 μg/mL). Selected sites show significant upregulation in non-sensitized tumor cells but not in sensitized cells (sensitized cells = orange line; sgTRAF2 + Birinipant). G CYLD knockout confers resistance to T cell-mediated killing in a WT background. BLM cells were transduced with indicated sgRNAs and subjected to a T cell cytotoxicity assay ± Birinapant (1 μg/mL). Viability was normalized to each condition’s untreated control and then further normalized to each treatment’s sgCtrl sample. Error bars represent standard deviation of biological replicates ( n = 3). Statistical analysis was performed with a two-way ANOVA with a Dunnet post-hoc test, using genotype and treatment as factors.

Article Snippet: D10 (ATCC) and BLM (ATCC) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma) and 2 mM glutamine.

Techniques: Phospho-proteomics, Co-Culture Assay, Knock-Out, Transduction, Cytotoxicity Assay, Control, Standard Deviation

Journal: Cell Death Discovery

Article Title: Inhibition of TBK1/IKKε mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing

doi: 10.1038/s41420-025-02841-x

Figure Lengend Snippet: A Flow cytometry plot of the competition assay of sgCtrl vs. sgTBK1 transduced D10 cells co-cultured with either MART-1 or Ctrl T cells for 3 days. B Quantification of the competition assay in ( A ) and additional conditions listed. Treatments were applied as follows: 1:8 T cell: tumor cell ratio; 1:2 NK cell: tumor cell ratio; 5 ng/ml INFgamma; 50 ng/ml TNF. Cells were sorted after 3 days. Error bars represent standard deviation between biological replicates ( n = 3). C Competition assay performed the same as in ( A , B ) but using BLM cell lines transduced with sgCtrl, sgTBK1, sgIKKε, sgTBK1+sgIKKε, or sgRNF31. Error bars represent standard deviation between biological replicates ( n = 3). D RIPK1 phosphorylation in the TNFR1 complex measured in sgCtrl and sgTBK1+sgIKKε melanoma cell lines following TNF-biotin AP-MS. Dots represent biological replicates ( n = 3 for BLM, n = 2 for D10). RIPK1 S320 phosphorylation was not detected in D10 cells. Phosphopeptide abundances were first normalized to endogenous RIPK1 protein levels within Complex I and then normalized to the maximum value for each site across experimental conditions. Abundace values are displayed as unitless normalized with the value of 1 representing the highest observed abundance. E Relative abundance of Complex I proteins in sgCtrl vs sgTBK1+sgIKKε tumor cells following TNF-biotin AP-MS. Horizontal line indicates a P value < 0.05, vertical lines indicate fold-change > 4, n = 3 biological replicates per condition.

Article Snippet: D10 (ATCC) and BLM (ATCC) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Sigma) and 2 mM glutamine.

Techniques: Flow Cytometry, Competitive Binding Assay, Cell Culture, Standard Deviation, Transduction, Phospho-proteomics, Protein-Protein interactions

Journal: BMC Pharmacology & Toxicology

Article Title: Human in vitro neuromuscular junction model to functionally dissect the pathogenic mechanism of anti-AChR autoantibody-positive myasthenia gravis

doi: 10.1186/s40360-025-01056-1

Figure Lengend Snippet: 3D compartmentalised design of human neuromuscular tissues on a microfluidic device. ( A ) The fabricated NeuroMuscle TM device uses the PDMS inlet microchannel to form a single NMJ microfluidic device, each composed of a muscle compartment with muscle fibre attaching pillar structures and the MN spheroids compartment. The muscle compartment has two medium reservoirs on each side. The MN compartment has a single medium reservoir. ( B ) Photo of the 3D NeuroMuscle TM microstructure. ( C ) Human iPSCs were seeded on an AggreWell™800 plate at Day 0 and MN spheroids were generated at Day 7. GCaMP6-transduced human skeletal myoblasts were injected into the muscle compartment of the NeuroMuscle TM device at day 0. Spontaneous self-organisation of myoblast into muscle bundle was observed after Day 5. A MN spheroid was introduced into the neuronal compartment and embedded in collagen gel. Thin neurite outgrowth was observed at Day 15 and many thick nerve bundles reached the muscle tissue at day 18, resulting in the formation of NMJs. Scale bars, 500 µm. ( D ) Differentiation of MN spheroids were characterised by the immunostaining of Tuj1, HB9, SMI-32, and VGLUT1. The pattern of a sarcomere structure stained by sarcomeric α-actinin and titin. SMI-32 staining showed that neurites from the motor neuron innervated muscle cells. AChR clusters are labelled with α-bungarotoxin (BTX; monochrome) and indicated by white arrows. Scale bars, 200 µm. ( E ) Schematic illustration of the differentiation and co-culture of the MN spheroid and muscle cells in a NeuroMuscle TM device. 3D, three-dimensional; AChR, acetylcholine receptor; BTX, α-bungarotoxin; D, day; DAPI, 4’,6-diamidino-2-phenylindole; GCaMP6, genetically encoded calcium indicator protein 6; iPSC, induced pluripotent stem cell; MN, motor neuron; NMJ, neuromuscular junction; PDMS, polydimethylsiloxane; SAA, serum amyloid a; Tuj1, class III beta-tubulin; VGLUT1, vesicular glutamate transporter 1

Article Snippet: Subsequently, the samples were incubated overnight with primary antibodies against class III beta-tubulin (1:200; Abcam, Cambridge, UK), HB9 (1:50; Santa Cruz, Dallas, TX, USA), SMI-32 (1:100; BioLegend, San Diego, CA, USA), vesicular glutamate transporter 1 (VGLUT1) (1:100; Abcam), sarcomeric α-actinin (1:100; Abcam), titin (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and C5b9 (1:500; BD Biosciences, Franklin Lakes, NJ, USA) in the blocking buffer.

Techniques: Generated, Injection, Immunostaining, Staining, Co-Culture Assay