Journal: Advanced Science

Article Title: Single‐Cell RNA Sequencing Reveals the Heterogeneity in Differentiation Trajectory and Tumor Microenvironment Leading to More Aggressive Phenotypes of Papillary Thyroid Cancer in Children and Young Adult Patients

doi: 10.1002/advs.202417672

Figure Lengend Snippet: Subtyping of cancer‐associated fibroblasts and their heterogeneity between CAYA and ADULT. A) UMAP plots for the four different CAF subtypes in papillary thyroid tumors (top left), and each cell colored for group (top right), tissue origin (bottom left). UMAP representations with cells colored by the expression level of marker genes of PTC. The black lines represent the spatial density of the cells expressing the given gene higher than the mean level of expression (bottom). B) Bubble plot showing top six differentially expressed genes in each CAF subtypes and patient. The size of the dot indicates the fraction of cells expressing a particular marker, and the intensity of the color represents the level of mean expression. Cellular phenotypes and age group are indicated left side the dot map. C) Bar plot showing the gene count for the most significantly upregulated GO BP pathways in each subtype, calculated through GSVA and Empirical Bayes Statistics for differential expression. D) Multiplex immunofluorescence staining showed major CAF clusters existed in PTC tissues. A pseudocolored image depicting different markers identified by mxIHC (colored as indicated in the key) and the results from histocytometry‐based cell classification (anti‐LAMP5 for emCAF_LAMP5, anti‐CD36 for lpmCAF_CD36, anti‐SMMC for myoCAF_MYH11, anti‐FBLN1 for iCAF_CFD). E) The pseudotime trajectory analysis of thyrocytes and fibroblasts inferred by Monocle 2. Each color represents one cell subtype (left), each point corresponds to one single cell (right). F) Trace plots of the EMT score along pseudotime separated by branch point. G) Shaded line plot indicating the expression levels of the EMT score (left) and TGF‐β (right) along the pseudotime in CAYA and adult patients. H) UMAP reflection of EMT scored by Ucell. I) Rank plot of TF (transcription factor) activities in the four CAF types, scored by pySCENIC. J) The binomial distribution heatmap of transcription factors of age‐specific transcription factors in CAYA and adults.

Article Snippet: A sequential application of antibodies against ANGPTL4 (NBP2‐80039, NOVUS, USA), LAMP5 (NBP1‐84246, NOVUS, USA), CD31 (BD, USA), CD36 (18836‐1‐AP, Proteintech, China), FBLN1 (NBP1‐84725, NOVUS, USA), MYH11 (21404‐1‐AP, Proteintech, China), VEGFC (22601‐1‐AP, Proteintech, China), VEGFR3 (20712‐1‐AP, Proteintech, China), and Pan‐CK (BD, USA) was performed, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies and subsequent tyramide signal amplification.

Techniques: Expressing, Marker, Quantitative Proteomics, Multiplex Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Aging-Associated Nox4 -Mediated Mitochondrial ROS and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1101/2025.07.09.664017

Figure Lengend Snippet: A : Representative fluorescence microscopy images and quantification of MitoSOX fluorescence in VSMCs treated with either a vehicle or 100 µM Ang II for 30 min. The scale bar is 2 µm. Data represent the integrated density of MitoSOX fluorescence (mean ± SEM, n = 8). B : The amount of 8-OHdG was measured by ELISA in total DNA from VSMCs treated with a vehicle or 100 µM Ang II for 24 h (mean ± SEM, n = 4). C : The oxygen consumption rate (OCR) was measured in VSMCs treated with a vehicle or Ang II for 24 h using an Agilent Seahorse XF96 analyzer (mean ± SEM, n = 12). D : Mitochondrial maximal respiration capacity was derived from OCR measurements in VSMCs treated with either a vehicle or Ang II for 24 h (mean ± SEM, n = 10). E : Mitochondrial spare capacity was also derived from OCR measurements in VSMCs treated with either a vehicle or Ang II for 24 h (mean ± SEM, n= 10). F : Flow cytometry analysis and quantification of MYH11 + TAGLN + cells in VSMCs treated with either a vehicle or Ang II for 24 h (mean±SEM, n=10). G : Flow cytometry analysis and quantification of CD11b + CD68 + cells in VSMCs treated with either a vehicle or Ang II for 24 h (mean ± SEM, n = 10).

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: (MYH11-FITC (Biorbyt, Durham, NC), CNN1-AlexaFluor 555 (Bioss), ACTA2-DyLight 405, TAGLN-PerCP (Novus Biologicals, Centennial, CO), TNFα-Brilliant Violet 510 (BioLegend), IL1b-APC-Cy7 (LifeSpan Bio, Newark, CA), and IL6-PerCP- eFluor 710 (Thermo Fisher).

Techniques: Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Aging-Associated Nox4 -Mediated Mitochondrial ROS and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1101/2025.07.09.664017

Figure Lengend Snippet: A : Western blot analysis of CGAS and STING expression in protein lysates from vehicle or Ang II-treated VSMCs isolated from wild-type, Nox4 TG, and Nox4 -/- mice. B : The quantification of CGAS protein expression in vehicle or Ang II-treated VSMCs. The data is fluorescence intensity fold change over vehicle- treated wild-type cells, adjusted for TUBB levels (mean ± SEM, n = 4). C : CGAMP levels were measured using ELISA in cell lysates from vehicle or Ang II-treated VSMCs (mean ± SEM, n = 4). D : The quantification of STING protein expression was analyzed with Western blot in vehicle or Ang II-treated VSMCs. Data are presented as fluorescence intensity fold change over vehicle-treated wild-type cells, adjusted for TUBB levels (mean ± SEM, n=4). E : Western blot analysis and quantification of CGAS protein levels in the abdominal aorta protein lysates from Apoe -/- , Nox4 TG/ Apoe -/- , and Nox4 -/- / Apoe -/- mice treated with Ang II. STING protein expression was quantified with Western blot in vehicle or Ang II-treated VSMCs. Data are presented as fluorescence intensity fold change over Apoe -/- mice, adjusted for ACTB levels (mean ± SEM, n = 4). F : Western blot analysis and quantification of STING and phospho-STING levels were performed on abdominal aorta protein lysates from Apoe -/- , Nox4 TG/ Apoe -/- , and Nox4 -/- / Apoe -/- mice treated with Ang II. Data are presented as fluorescence intensity fold change over Apoe -/- mice, adjusted for ACTB levels (mean ± SEM, n = 4). G & H : Representative fluorescence microscopy images and quantification of CGAS ( G ) and STING ( H ) expression in human control aorta and AAA frozen sections. These sections were stained for immunoreactive CGAS ( G ) or STING ( H ) (red) and MYH11 (green), with DAPI (blue) used for counterstaining. The scale bar is 100 µm. Data are presented as fluorescence integrated density (mean ± SEM, n = 9).

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: (MYH11-FITC (Biorbyt, Durham, NC), CNN1-AlexaFluor 555 (Bioss), ACTA2-DyLight 405, TAGLN-PerCP (Novus Biologicals, Centennial, CO), TNFα-Brilliant Violet 510 (BioLegend), IL1b-APC-Cy7 (LifeSpan Bio, Newark, CA), and IL6-PerCP- eFluor 710 (Thermo Fisher).

Techniques: Western Blot, Expressing, Isolation, Fluorescence, Enzyme-linked Immunosorbent Assay, Microscopy, Control, Staining

Journal: bioRxiv

Article Title: Aging-Associated Nox4 -Mediated Mitochondrial ROS and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1101/2025.07.09.664017

Figure Lengend Snippet: A & B : Flow cytometry analysis of single-cell suspension from the abdominal aorta of mice treated with Ang II. Panel A presents the quantification of MYH11 + cells, while Panel B shows the quantification of CD45 + CD68 + CD11b + cells. The data represent the cell fraction of all aortic cells (mean ± SEM, n=6). C : Flow cytometry analysis and quantification of abdominal aorta single-cell suspension from Ang II-treated mice showing the proportion of MYH11 + cells expressing macrophage markers. This data shows the fraction of CD68 + CD11b + cells as a percentage of MYH11 + cells (mean ± SEM, n = 6). D : A t-SNE clustering analysis of flow cytometry data from the abdominal aorta of Ang II-treated mice was performed. MYH11 + cells were clustered based on the expression of CNN1, ACTA2, TAGLN, CD45, CD68, CD11b, CD38, TNFα, IL1b, and IL6, resulting in distinct clusters presented on the t-SNE plot. E : A heat map representation illustrates the relative expression of SMC, macrophage, and inflammatory markers based on the mean fluorescence intensity for each cluster. F & H : The relative abundance of distinct clusters as a proportion of MYH11 + cells (mean ± SEM, n = 6).

Article Snippet: Samples were incubated in 200 μL FACS buffer with a mix of intracellular marker antibodies: (MYH11-FITC (Biorbyt, Durham, NC), CNN1-AlexaFluor 555 (Bioss), ACTA2-DyLight 405, TAGLN-PerCP (Novus Biologicals, Centennial, CO), TNFα-Brilliant Violet 510 (BioLegend), IL1b-APC-Cy7 (LifeSpan Bio, Newark, CA), and IL6-PerCP- eFluor 710 (Thermo Fisher).

Techniques: Flow Cytometry, Suspension, Expressing, Fluorescence