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: Lysed mouse VSMCs were tested for 8-OHdG expression using OxiSelectTM Oxidative DNA Damage ELISA Kit (Thermo Fisher) and for 2’3’-cGAMP using 2’3’- cGAMP EIA Kit (Cayman Chemical, Ann Arbor, MI), following the manufacturer’s protocols.

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: Lysed mouse VSMCs were tested for 8-OHdG expression using OxiSelectTM Oxidative DNA Damage ELISA Kit (Thermo Fisher) and for 2’3’-cGAMP using 2’3’- cGAMP EIA Kit (Cayman Chemical, Ann Arbor, MI), following the manufacturer’s protocols.

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