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: bioRxiv

Article Title: Cachexia and fibrosis are costs of chronic IL-1R-mediated disease tolerance in T. gondii infection

doi: 10.1101/783316

Figure Lengend Snippet: A-C Western blot for alpha smooth muscle actin (α-SMA) or GAPDH on liver (A) , quadriceps (B) , and vWAT (C) tissue lysate at 9 wpi. Representative of at least 3 independent experiments. Each lane represents an individual mouse. D-F , Picrosirius red staining on formalin-fixed paraffin-embedded liver (D) , gastrocnemius (E) , or vWAT (F) using polarized light at 9 weeks post-infection. Representative images shown at left. Picrosirius red staining was quantified in ImageJ (right) and pixel density represented as % of each field of view. Each point represents a field of view (N=3 mice per group, 5-10 fields of view per mouse were quantified). Scale bars represent 50 μm. D-E are representative of at least 2 experiments and (F) is pooled between two independent experiments. Error bars are standard error of the mean. Statistical outliers were removed using the ROUT method (Q=1%), in D-F. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Student’s T test.

Article Snippet: The next morning, samples were washed 3 times in PBS/0.1% Triton-X and were incubated in secondary antibody (Novus NBP1-75607 donkey anti-goat Dylight 594, 1:250 and LifeTech A21206 donkey anti-rabbit AF488, 1:200), and the directly conjugated α-SMA primary (Novus Biologicals NBP2-34760APC, 1:400) for 1 hour at room temperature, diluted in PBS/0.1% Triton-X.

Techniques: Western Blot, Staining, Formalin-fixed Paraffin-Embedded, Infection

Journal: bioRxiv

Article Title: Cachexia and fibrosis are costs of chronic IL-1R-mediated disease tolerance in T. gondii infection

doi: 10.1101/783316

Figure Lengend Snippet: A-C Western blot for alpha smooth muscle actin (α-SMA) or GAPDH on liver (A), vWAT (B), and quad (C) tissue lysate at 2 or 9 wpi. Representative of at least 2 independent experiments. Each lane represents an individual mouse. D-F Picrosirius red staining on formalin-fixed paraffin-embedded liver (D), gastrocnemius (E), or vWAT (F) using brightfield (D) or polarized light (E-F) at 9 weeks post-infection. Picrosirius red staining was quantified in ImageJ (below) and pixel density represented as % of each field of view. N=3 mice per group, 5-10 fields of view per mouse were quantified Representative of 2 experiments. Error bars are standard error of the mean. Scale bars represent 50 μm. Statistical outliers were removed using the ROUT method (Q=1%), in E and F. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 by unpaired Student’s T test with Holm-Sidak method to correct for multiple comparisons.

Article Snippet: The next morning, samples were washed 3 times in PBS/0.1% Triton-X and were incubated in secondary antibody (Novus NBP1-75607 donkey anti-goat Dylight 594, 1:250 and LifeTech A21206 donkey anti-rabbit AF488, 1:200), and the directly conjugated α-SMA primary (Novus Biologicals NBP2-34760APC, 1:400) for 1 hour at room temperature, diluted in PBS/0.1% Triton-X.

Techniques: Western Blot, Staining, Formalin-fixed Paraffin-Embedded, Infection

Journal: bioRxiv

Article Title: Cachexia and fibrosis are costs of chronic IL-1R-mediated disease tolerance in T. gondii infection

doi: 10.1101/783316

Figure Lengend Snippet: A , Cytokines in liver lysates from mice 9 wpi were measured by ELISA. Data are presented as fold change relative to the mean of uninfected levels. N=4-13 mice per group, pooled from two independent experiments. B-C , Immunofluorescence labeling of nuclei (DAPI white) IL-1α (green), CD45 (red) and collagen1α1 (blue) ( B ) nuclei (DAPI white) α-smooth muscle actin (green), IL-1R (red) and collagen1α (blue) in the liver of uninfected or 9 wpi WT or IL-1R -/- mice ( C ). Inset, arrow head represents α-smooth muscle actin, IL-1R co-staining cells (arrow heads). ( B-C ) represent maximum intensity projections of 9-13 μm thick z-stacks. Scale bar represents 50 μm. Error bars are standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Student’s T test with Holm-Sidak method to correct for multiple comparisons.

Article Snippet: The next morning, samples were washed 3 times in PBS/0.1% Triton-X and were incubated in secondary antibody (Novus NBP1-75607 donkey anti-goat Dylight 594, 1:250 and LifeTech A21206 donkey anti-rabbit AF488, 1:200), and the directly conjugated α-SMA primary (Novus Biologicals NBP2-34760APC, 1:400) for 1 hour at room temperature, diluted in PBS/0.1% Triton-X.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Labeling, Staining

Journal: bioRxiv

Article Title: Cachexia and fibrosis are costs of chronic IL-1R-mediated disease tolerance in T. gondii infection

doi: 10.1101/783316

Figure Lengend Snippet: A-C , MEF cells were incubated with media, 10ng/mL IL-1α or 5 ng/mL TGFβ-1 for 48 hours. After fixation, MEFs were stained for F-actin, alpha-smooth muscle actin (α-SMA), phalloidin and nuclei and cell spreading was quantified in (B) , and levels of α-SMA expression were quantified in terms of pixels/cell (C). D-F , Primary hepatic stellate cells (HSCs) were isolated from uninfected mouse livers, and FACS sorted based on endogenous retinoid fluorescence. Retinoid fluorescence was validated by UV photobleaching ( D ). E-F , HSCs were seeded onto 4kPa hydrogels coated with 10ug/mL of fibronectin and cultured with 10ng/mL IL-1α, 10 ng/mL TGF-β, or media alone for 48hrs and then fixed and stained for F-actin and α-SMA and imaged by confocal microscopy. Scale bar represents 50 μm. Total cell area quantified in ( F ) Error bars are standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 by unpaired Student’s T test.

Article Snippet: The next morning, samples were washed 3 times in PBS/0.1% Triton-X and were incubated in secondary antibody (Novus NBP1-75607 donkey anti-goat Dylight 594, 1:250 and LifeTech A21206 donkey anti-rabbit AF488, 1:200), and the directly conjugated α-SMA primary (Novus Biologicals NBP2-34760APC, 1:400) for 1 hour at room temperature, diluted in PBS/0.1% Triton-X.

Techniques: Incubation, Staining, Expressing, Isolation, Fluorescence, Cell Culture, Confocal Microscopy

Journal: Annals of Gastroenterological Surgery

Article Title: Intratumoral Fusobacterium nucleatum Drives Cancer‐Associated Fibroblasts Enrichment and Immune Exclusion in Esophageal Squamous Cell Carcinoma

doi: 10.1002/ags3.70116

Figure Lengend Snippet: Histopathological assessment of CAFs in ESCC. (a) Representative FISH images of ESCC tissues showing signals for F. nucleatum (red; FUS664), all bacteria (green; EUB338), and nuclei (blue; DAPI). Positive F. nucleatum signals were observed in cases that were identified as F. nucleatum –positive by qPCR. All images were acquired at ×200 magnification. (b) Representative FISH images of ESCC tissues obtained by laser scanning confocal microscopy, demonstrating intracellular signals of Fusobacterium nucleatum (red; FUS664) within tumor cells. Nuclei were counterstained with DAPI (blue). Images were acquired at ×400 magnification. (c) Representative FISH images of ESCC tissues showing signals for F. nucleatum (red; FUS664), all bacteria (green; EUB338), and nuclei (blue; DAPI). F. nucleatum signals were observed within tumor cells located in regions enriched with α‐SMA–positive CAFs. The boxed areas indicate regions of interest, imaged at ×200 magnification. ** p < 0.01.

Article Snippet: We used monoclonal mouse anti α‐SMA antibody (1:250; #56856; Cell Signal Technology) and RelA (1:800; #8242; Cell Signal Technology) as the primary antibody, following the manufacturer's protocols.

Techniques: Bacteria, Confocal Microscopy

Journal: Annals of Gastroenterological Surgery

Article Title: Intratumoral Fusobacterium nucleatum Drives Cancer‐Associated Fibroblasts Enrichment and Immune Exclusion in Esophageal Squamous Cell Carcinoma

doi: 10.1002/ags3.70116

Figure Lengend Snippet: Immunohistochemical analysis of NF‐κB activation and its association with F. nucleatum and CAFs in ESCC. (a) The proportion of NF‐κB–positive tumors was significantly higher in F. nucleatum –positive cases than in F. nucleatum –negative cases. (b) Representative immunohistochemical staining on adjacent serial sections of ESCC tissues showing stromal α‐SMA expression and nuclear RelA localization in tumor cells. These signals were observed in close proximity. Images were acquired at ×200 magnification. (c) Dual positivity for stromal α‐SMA and NF‐κB–positive in tumor cells was significantly enriched in F. nucleatum –positive tumors compared to F. nucleatum –negative tumors. (d) Summary of the results. F. nucleatum contributes to the progression of ESCC by inducing NF‐κB–mediated inflammatory signaling in tumor cells and promoting the activation of CAFs. ** p < 0.01.

Article Snippet: We used monoclonal mouse anti α‐SMA antibody (1:250; #56856; Cell Signal Technology) and RelA (1:800; #8242; Cell Signal Technology) as the primary antibody, following the manufacturer's protocols.

Techniques: Immunohistochemical staining, Activation Assay, Staining, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Aldolase a coordinates macropinocytic nutrient scavenging and lysosomal degradation in lung cancer by interacting with V-ATPase

doi: 10.1186/s12964-025-02591-4

Figure Lengend Snippet: Targeting ALDOA impairs lysosomal proteolysis and tumor growth in vivo. A Immunoblot analysis confirming the effect of Aldolase A (ALDOA)-targeting sgRNA on protein levels of ALDOA in A549 cells. B Effects of bovine serum albumin (BSA) supplementation on proliferation of sgALDOA cells under leucine-replete and leucine-deprived conditions. C , D Representative gross images ( C ) and growth curves ( D ) of xenografted tumors derived from A549 control or sgALDOA cells, treated with or without the macropinocytosis inhibitor 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) ( N = 6 per group). E Representative immunofluorescence images of Lysosome (red) and ALDOA (green) in tumor tissues from xenografted A549 models. Arrows indicate colocalized puncta. F Representative images of DQ-BSA fluorescence (left panel) and quantification of DQ-BSA fluorescence intensity (right panel) in tumor tissue formed after subcutaneous injection of Ctrl or sgALDOA A549 cells, with or without EIPA treatment. G Representative histological (H&E) and immunohistochemical images of Ki67 and p-S6 staining (left panel), and quantification of Ki67 and p-S6 expression scores (right panel), in xenograft tumor sections. H Representative immunofluorescence images of ALDOA (green) and α-smooth muscle actin (α-SMA, red) in xenograft tumor sections (left panel), and quantification of ALDOA fluorescence in α-SMA–negative cells (right panel). All experimental data were verified in at least six independent experiments. Scale bar, 10 μm. Data are presented as the mean ± SEM. ns., not significant, ** p < 0.01, and *** p < 0.001

Article Snippet: For tumor tissue staining, deparaffinized samples were incubated with anti-ALDOA (Proteintech) and anti-LAMP1 (Santa Cruz) or anti-ALDOA (Santa Cruz) and anti-α-smooth muscle actin (α-SMA) (Cell Signaling).

Techniques: In Vivo, Western Blot, Derivative Assay, Control, Immunofluorescence, Fluorescence, Injection, Immunohistochemical staining, Staining, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Design of RGD-functionalized GSH-responsive pegylated polymeric protacs for selective BRD4 degradation and EndMT-driven cardiac fibrosis inhibition

doi: 10.1186/s12951-026-04036-7

Figure Lengend Snippet: RGD-PEG-MZ1 exerted a profound inhibitory effect on the EndMT in vitro. ( A-D ) The expression of Collagen-I, Fibronectin, VE-cadherin, α-SMA and FSP1 was assessed using western blotting (WB) in HUVECs and HMEC-1. ( E ) Immunofluorescence staining for α-SMA (green) and VE-cadherin (green) in HUVECs. Scale bar, 100 μm. The data are presented as the Mean ± SD of three independent experiments. # p < 0.05 and ## p < 0.01 vs. NC group; ns, p > 0.05 vs. control group; * p < 0.05 and ** p < 0.01 vs. control group (one-way ANOVA followed by Bonferroni post hoc test)

Article Snippet: The slides were initially stabilized with a 5% goat serum solution, then exposed to primary antibodies—α-SMA (Proteintech, 14395-1-AP), Collagen-I (Proteintech, 14695-1-AP), CD31 (Proteintech, 66065-2-Ig), Raf1 (Abcam, ab181115), Raf1 (phospho S259) (Abcam, ab173539), ERK1 + ERK2 (Abcam, ab184699) and ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) (Abcam, ab278538)—and subsequently incubated at 4 °C for an extended period.

Techniques: In Vitro, Expressing, Western Blot, Immunofluorescence, Staining, Control