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

Journal: bioRxiv

Article Title: Injured endocardium obtains characteristics of haemogenic endothelium during adult zebrafish heart regeneration

doi: 10.1101/2024.12.18.629122

Figure Lengend Snippet: A-B, Fluorescent imaging of live sections from injured endocardium at 3dpci over 15h shows endocardial cells became rounder, detached from the cell layer and disappeared out of view (arrowheads). C, Evidence of kdrl -positive cell detachment from the endothelium in a coronary vessel (arrowhead). D, Immunostaining of YFP and MF20 at 3dpci and 7dpci in Tg(fli1a-creERT2);Tg(ubiCSY) zebrafish. The endocardium is labelled by YFP with cells in the injured area showing different morphology from the rest of the ventricle. Arrowhead at 7dpci points to YFP-positive lineage traced circulating blood cell. E, Staining of YFP and lyC reveals co-localisation of YFP and lyC expression, indicating that endocardially traced cells obtain characteristics of leukocytes. F, Immunofluorescence images for YFP and kdrl at 3dpci in Tg(fli1a-creERT2);Tg(ubiCSY) fish shows that while most YFP-labelled cells still express kdrl mRNA indicating that these cells are endocardial, the more rounded YFP-positive endocardial cells as well as the circulating cells do not express kdrl mRNA (arrowhead). These data indicate that rounding up endocardial cell lose their endocardial identity when they undergo EHT. This data also shows that kdrl is absent from the endocardially derived circulating cells. Scale bar: 100µm.

Article Snippet: The following primary antibodies were used: chicken polyclonal against Green Fluorescent Protein (GFP, 1:200, Aves Lab, GFP-1020), mouse monoclonal against mCherry (clone 1C51, 1:200, Abcam, ab125096), Proliferating Cell Nuclear Antigen (PCNA, clone PC10, 1:200, Dako Cytomation, M0879), Myosin Heavy Chain (MF20, 1:50, HSHB AB-2147781), cardiac Troponin-T (cTnT, 1:500, Abcam, ab33589) and rabbit polyclonal against Lysozyme (LyC, 1:200, Anaspec, AS-55633), smooth muscle Myosin heavy chain 11 (Myh11, 1:200, Abcam, ab125884).

Techniques: Imaging, Immunostaining, Staining, Expressing, Immunofluorescence, Derivative Assay

Journal: Aging (Albany NY)

Article Title: Intracranial aneurysm circulating exosome-derived LncRNA ATP1A1-AS1 promotes smooth muscle cells phenotype switching and apoptosis

doi: 10.18632/aging.205821

Figure Lengend Snippet: Overexpression of ATP1A1-AS1 promotes VSMC phenotype switching and apoptosis. ( A ) ATP1A1-AS1 mimics were used to overexpress ATP1A1-AS1 in VSMCs. The quantification of ATP1A1-AS1 expression was performed using qRT-PCR. ( B , C ) WB were performed to quantify the protein expression levels of contractile markers ( B ) and MMP-2 and MMP-9 ( C ) after overexpression of ATP1A1-AS1 in VSMCs. ( D , E ) WB was performed to detect contraction markers ( D ) and MMP-2 and MMP-9. ( E ) Protein expression levels after overexpression of ATP1A1-AS1 in pathological VSMCs, simulated using 400 μM hydrogen peroxide. ( F – I ) Quantification of protein expression levels. ( J ) TUNEL assay used to detect apoptosis after overexpression of ATP1A1-AS1 in pathological VSMCs. ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The membranes were then blocked with protein-free rapid blocking buffer (Meilun, Dalian, China) for 10 minutes, and probed with the following primary antibodies: anti-α-SMA (BM0002, Boster, Wuhan, China), anti-CNN1 (abs171608, Absin, Shanghai, China), anti-MYH11 (K002095P, Solarbio, Beijing, China), anti-MMP2 (A00286-2, Boster, Wuhan, China), and anti-MMP9 antibodies (BM4089, Boster, Wuhan, China).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, TUNEL Assay

Journal: Aging (Albany NY)

Article Title: Intracranial aneurysm circulating exosome-derived LncRNA ATP1A1-AS1 promotes smooth muscle cells phenotype switching and apoptosis

doi: 10.18632/aging.205821

Figure Lengend Snippet: Knockdown of ATP1A1-AS1 inhibits VSMC phenotype switching and apoptosis. ( A ) ATP1A1-AS1 siRNA was used to knockdown ATP1A1-AS1 in VSMCs. The expression level of ATP1A1-AS1 was determined using qRT-PCR. ( B , C ) WB was performed to detect contraction markers proteins ( B ) and MMP-2 and MMP-9 ( C ) expression levels after knockdown of ATP1A1-AS1 in pathological VSMCs, simulated using 400 μM hydrogen peroxide. ( D , E ) Quantification of protein expression levels. ( F ) TUNEL assay used to detect apoptosis after knockdown of ATP1A1-AS1 in pathological VSMCs ( * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The membranes were then blocked with protein-free rapid blocking buffer (Meilun, Dalian, China) for 10 minutes, and probed with the following primary antibodies: anti-α-SMA (BM0002, Boster, Wuhan, China), anti-CNN1 (abs171608, Absin, Shanghai, China), anti-MYH11 (K002095P, Solarbio, Beijing, China), anti-MMP2 (A00286-2, Boster, Wuhan, China), and anti-MMP9 antibodies (BM4089, Boster, Wuhan, China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, TUNEL Assay