Journal: International Journal of Molecular Sciences

Article Title: A Simple Ionic-Gelation Method for Chitosan Nanoparticle Synthesis and Standardized Protocols for Biological Safety Assessment: Antibacterial Activity, Phytotoxicity, and Biocompatibility

doi: 10.3390/ijms27083673

Figure Lengend Snippet: Cell viability of HDF cells treated with Ch NPs at concentrations of 2.5–160 µg/mL for 24 h. Viability was determined using the MTT assay and expressed as mean ± SE (n = 3). Different letters indicate statistically significant differences ( p < 0.05).

Article Snippet: The biocompatibility of Ch NPs was evaluated using primary HDF (ATCC ® PCS-201-012TM [ ], American Type Culture Collection, Manassas, VA, USA) and HaCaT cells (Cat. No. T0020001, AddexBio, San Diego, CA, USA).

Techniques: MTT Assay

Journal: Cells

Article Title: Fixed-Bed Bioreactor Culture Enhances Yield and Reparative Properties of hTERT Mesenchymal Stem Cell Extracellular Vesicles

doi: 10.3390/cells15070654

Figure Lengend Snippet: EV function and signaling activity. ( a ) Primary keratinocytes were exposed to IR and treated with EVs from either flask or bioreactor cultures. Cell viability was assessed at 24, 48, and 72 h using the CellTiter-Glo assay. ( b ) Non-irradiated keratinocytes were treated with EVs from flask or bioreactor cultures, and viability was assessed after 72 h using the same assay. * = p < 0.05; *** = p < 0.001; **** = p < 0.0001. ( c ) Western blot of cell cycle proteins associated with hTERT MSC EVs produced from both the bioreactor and flask-based cultures. ( d ) In vitro kinase assay of EV-associated CDK2. EVs were immunoprecipitated with CDK2 antibodies or IgG control and kinase activity was assessed using Histone H1 as substrate.

Article Snippet: Normal primary epidermal keratinocytes (ATCC ® PCS-200-011TM) were maintained in Dermal Cell Basal Medium (ATCC ® PCS-200-030TM) supplemented with Keratinocyte Growth Kit (ATCC ® PCS-200-040TM).

Techniques: Activity Assay, Glo Assay, Irradiation, Western Blot, Produced, In Vitro, Kinase Assay, Immunoprecipitation, Control

Journal: Cell Death Discovery

Article Title: Trifluoperazine causes mast cell apoptosis through a secretory granule-mediated pathway

doi: 10.1038/s41420-026-03122-x

Figure Lengend Snippet: A Human lung smooth muscle cells (HLSMCs), lung fibroblasts (HLFs), and small airway epithelial cells (HSAECs) were treated with TFP at the indicated concentrations for 24 h. Human peripheral blood eosinophils and neutrophils were treated with TFP for 2 h. Cell viability was assessed by staining the cells with Annexin V (AnnV) and DRAQ7. Viable cells, AnnV − DRAQ7 − ; apoptotic cells, AnnV + DRAQ7 − ; necrotic/late apoptotic cells, AnnV + DRAQ7 + . HSAECs, n = 6 from three independent experiments; HLSMCs, eosinophils, neutrophils, n = 4 from four independent experiments; HLFs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA for HLSMCs, HLFs; Kruskal-Wallis for HSAECs; Friedman test for eosinophils, neutrophils). B – C Bone marrow-derived MCs (BMMCs) and peritoneal cell-derived MCs (PCMCs) treated under the same conditions as in ( A ) for 24 h. BMMCs, n = 5 from two independent experiments; PCMCs, n = 3 from one individual experiment representative of three independent experiments (One-way ANOVA). Untreated (control) cells were used for statistical comparisons to all other groups in all figures. The bar charts show mean values + SEM or median + interquartile range. * P < 0.05; ** P < 0 .01; **** P < 0.0001. D Effect of TFP on DNA degradation. MCs were preincubated with bafilomycin A1 (Baf A1) (20 nM) for 2 h followed by treatment with TFP (10 μΜ) for 2 h. DNA was extracted from MCs and fragmentation was assessed by agarose gel electrophoresis. St standard marker.

Article Snippet: Primary human lung fibroblasts (HLFs) (PCS-201-013) and primary human lung smooth muscle cells (HLSMCs)(PCS-130-010) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Control, Agarose Gel Electrophoresis, Marker

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Endothelial cell-derived exosomes influence vascular smooth muscle cell phenotype and calcification-related gene expression. HAVSMCs were incubated for 8 days with 10 µg/mL exosomes derived from endothelial cells (ECs) in ECM (control), TNFα, TGFβ, or varying concentrations of TMAO (1–100 μM). ( A – D ) qPCR analysis of osteogenic markers RUNX2 and OPN, confirming transcriptional reprogramming toward an osteoblast-like phenotype. ( E ) TNAP (Tissue Non-Specific Alkaline Phosphatase) involved in vascular calcification and osteogenic transformation of VSMCs. Data are presented as mean ± SD from four independent biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. SMCM control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Derivative Assay, Gene Expression, Incubation, Control, Transformation Assay

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Differential effects of endothelial cell-derived exosomes on calcification of HAVSMCs, assessed by Alizarin Red staining. ( A – G ) Representative images of Alizarin Red staining in HAVSMCs after 8 days of culture with 10 µg/mL endothelial cell-derived exosomes (EC-EXOs) obtained from endothelial cell maintenance medium (ECM EC EXO), TNFα-stimulated EC exosomes (TNFα EC EXO), TGFβ-stimulated EC exosomes (TGFβ EC EXO), TMAO-treated EC exosomes (1 µM, 10 µM, and 50 µM TMAO EC EXO), and control smooth muscle cell medium (SMCM). ( H ) Quantification of Alizarin Red stain intensity was normalized to total protein concentration. Data are presented as mean ± SD from four independent biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05 vs. SMCM control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Derivative Assay, Staining, Control, Protein Concentration

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: β-catenin inhibition attenuates endothelial exosome-induced β-catenin activation in HAVSMCs. ( A , C ) Representative Western blot images showing non-phosphorylated (active) β-catenin protein expression in human aortic vascular smooth muscle cells (HAVSMCs) treated with endothelial cell-derived exosomes (EC-EXOs) obtained from TNFα-, TGFβ-, or TMAO-stimulated endothelial cells, in the presence or absence of the β-catenin transcriptional inhibitor ICG-001 for 8 days. β-actin was used as a loading control. ( B , D ) Quantitative densitometric analysis demonstrates a significant increase in β-catenin protein levels following EC-EXO treatment, which was markedly reduced upon β-catenin inhibition with ICG-001. Protein expression levels were normalized to β-actin and expressed as fold change relative to vehicle-treated controls. Data are presented as mean ± standard deviation (SD) from three independent biological replicates. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test to assess differences between EC-EXO treatment groups and the effect of β-catenin inhibition. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Derivative Assay, Control, Standard Deviation

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: β-catenin inhibition suppresses endothelial exosome-induced osteogenic gene expression in HAVSMCs. ( A – E ) Quantitative real-time PCR analysis of osteogenic gene expression in HAVSMCs treated with endothelial cell-derived exosomes (EC-EXOs) from TNFα-, TGFβ-, or TMAO-stimulated endothelial cells, in the presence of the β-catenin inhibitor ICG-001. Relative mRNA expression levels of ( A ) SM22A, ( B ) αSMA, ( C ) RUNX2, ( D ) osteopontin (OPN), and ( E ) tissue-nonspecific alkaline phosphatase (TNAP) were normalized to housekeeping genes and expressed relative to vehicle-treated control cells (0.1% v / v DMSO). EC-EXO co-treatment with ICG-001 significantly attenuated the expression of RUNX2, OPN, and TNAP, indicating that β-catenin signaling is required for endothelial exosome-induced osteogenic reprogramming of HAVSMCs. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was assessed using one-way ANOVA, followed by post-hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CTL vehicle.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Inhibition, Gene Expression, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Control

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Uptake kinetics of MemBright-labeled endothelial cell-derived exosomes by HAVSMC. Representative confocal microscopy images showing the time-dependent uptake of MemBright-labeled endothelial cell-derived exosomes by human aortic vascular smooth muscle cells (HAVSMCs). ( A ) HAVSMCs treated with control endothelial cell-derived exosomes (CTL EC EXO). ( B ) HAVSMCs treated with exosomes derived from endothelial cells exposed to 50 µM TMAO (TMAO EC EXO). Exosomes were labeled with MemBright (green), and cell nuclei were counterstained with Hoechst (blue). Images were acquired immediately after exosome addition (T = 0 h) and after 1, 3, and 4 h of incubation. Merged images illustrate progressive internalization and intracellular accumulation of exosomes over time, with 20× objective. All images were captured using a Leica confocal laser scanning microscope under identical acquisition settings. Scale bar: 194 µm.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Labeling, Derivative Assay, Confocal Microscopy, Control, Incubation, Laser-Scanning Microscopy

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: miR-222-3p overexpression promotes osteogenic signaling in HAVSMCs through activation of β-catenin pathway. ( A ) Quantitative PCR analysis confirming successful transfection of HAVSMCs with miR-222-3p mimic compared with the results for scrambled mimic control. Relative miR-222-3p expression levels were normalized to miR5S and expressed as fold change. ( B – F ) Quantitative PCR analysis of gene expression levels of RUNX2, OPN and TNAP in HAVSMCs after miR-222-3p mimic transfection for 48 h. ( G ) Representative Western blot images showing β-catenin protein expression in HAVSMCs following transfection with scrambled mimic or miR-222-3p mimic. ( H ) Quantitative densitometric analysis of protein expression levels of β-catenin protein expression levels were normalized to housekeeping protein and expressed relative to scrambled control. Data are presented as mean ± SD from independent biological replicates. Statistical significance was determined using unpaired two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. scrambled mimic control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Over Expression, Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Control, Expressing, Gene Expression, Western Blot, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: High-Content Imaging and Machine Learning Classify Phenotypical Change in Coronary Artery Endothelial Cells Caused by BPS

doi: 10.3390/ijms27073259

Figure Lengend Snippet: Representative high-content microscopy images of human coronary artery endothelial cells (HCAEC) exposed to vehicle control (CTRL) or 0.1 µM Bisphenol S (BPS) for 96 h and stained using the PhenoVue Cell Painting assay. For each condition, a representative field acquired at 40× magnification and a higher-magnification inset are shown. Rows correspond to the individual fluorescence channels: Hoechst 33342 (nuclei), PhenoVue Fluor 488 Concanavalin A (endoplasmic reticulum and intracellular membranes), PhenoVue 512 nucleic acid stain (RNA/nucleoli), PhenoVue Fluor 555 wheat germ agglutinin (plasma membrane), PhenoVue 641 mitochondrial stain (mitochondria), and the merged image. White boxes represent the part of the image used for the related inset. Scale bar: 50 µm, 40× objective.

Article Snippet: Primary human coronary artery endothelial cells (HCAEC; ATCC ® PCS-100-020TM, Innovation, VA, USA) were cultured according to the supplier’s recommendations.

Techniques: Microscopy, Control, Staining, Fluorescence, Clinical Proteomics, Membrane