rabbit anti 8 ohdg  (Bioss)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Aging‐Associated Nox4 ‐Mediated Mitochondrial Reactive Oxygen Species and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1161/JAHA.125.044949

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 square root‐transformed 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 OCR was measured in VSMCs treated with a vehicle or Ang II for 24 h using an Agilent Seahorse XF96 analyzer (mean±SEM, n=10). 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). 8‐OHdG indicates 8‐hydroxy‐2′‐deoxyguanosine; Ang II, angiotensin II; FCCP; MitoSOX, mitochondrial superoxide; MYH11, myosin heavy chain 11; Nox4 −/− , Nox4 knockout mice; Nox4 TG, Nox4 transgenic mice; OCR, oxygen consumption rate; Rot+AA, Rotenone/Antimycin A; TAGLN, transgelin; and VSMC, vascular smooth muscle cell.

Article Snippet: The immunostaining was conducted using the following antibodies: NOX4, TOMM20 (translocase of outer mitochondrial membrane 20)‐AlexaFluor488, IL1b (Abcam: ab109225, ab205486, ab254360); FGA, DNASE2 (deoxyribonuclease II; Thermo Fisher: PA5‐141128, PA5‐115136); 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), ATP5G (ATP synthase membrane subunit C locus1)‐AlexaFluor488, CD68‐AlexaFluor488, CD68‐Cy3, MYH11 (myosin heavy chain 11)‐AlexaFluor488 (Bioss, Woburn, MA: bs‐1278R, bs‐12547R‐A488, bs‐12547R‐A488, bs‐0649R‐Cy3, bs‐1956R‐A488); ACTA2‐FITC (actin alpha 2, smooth muscle), ACTA2‐Cy3 (Sigma‐Aldrich, St. Louis, MO: F3777, C6198); CD11b (Abnova, Walnut, CA: PAB12136 ); cyclic GMP‐AMP synthase (cGAS), STING (stimulator of interferon genes), and IL6 (interleukin‐6; Cell Signaling Technology, Danvers, MA: 15102S, 13647S, 12912S).

Techniques: Fluorescence, Microscopy, Transformation Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Flow Cytometry, Knock-Out, Transgenic Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Aging‐Associated Nox4 ‐Mediated Mitochondrial Reactive Oxygen Species and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1161/JAHA.125.044949

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 are expressed as fold change in fluorescence intensity normalized to vehicle‐treated wild‐type cells, adjusted for TUBB levels (mean±SEM, n=4). C, CGAMP levels were measured using EIA 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 log‐transformed fluorescence integrated density (mean±SEM, n=9). AAA indicates abdominal aortic aneurysm; ACTB, actin beta; Ang II, angiotensin II; Apoe −/− , apolipoprotein E knockout mice; cGAS, cyclic GMP‐AMP synthase; MYH11, myosin heavy chain 11; Nox4 TG, Nox4 transgenic mice; STING, stimulator of interferon gene; TUBB, tubulin beta; and VSMC, vascular smooth muscle cell.

Article Snippet: The immunostaining was conducted using the following antibodies: NOX4, TOMM20 (translocase of outer mitochondrial membrane 20)‐AlexaFluor488, IL1b (Abcam: ab109225, ab205486, ab254360); FGA, DNASE2 (deoxyribonuclease II; Thermo Fisher: PA5‐141128, PA5‐115136); 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), ATP5G (ATP synthase membrane subunit C locus1)‐AlexaFluor488, CD68‐AlexaFluor488, CD68‐Cy3, MYH11 (myosin heavy chain 11)‐AlexaFluor488 (Bioss, Woburn, MA: bs‐1278R, bs‐12547R‐A488, bs‐12547R‐A488, bs‐0649R‐Cy3, bs‐1956R‐A488); ACTA2‐FITC (actin alpha 2, smooth muscle), ACTA2‐Cy3 (Sigma‐Aldrich, St. Louis, MO: F3777, C6198); CD11b (Abnova, Walnut, CA: PAB12136 ); cyclic GMP‐AMP synthase (cGAS), STING (stimulator of interferon genes), and IL6 (interleukin‐6; Cell Signaling Technology, Danvers, MA: 15102S, 13647S, 12912S).

Techniques: Western Blot, Expressing, Isolation, Fluorescence, Microscopy, Control, Staining, Transformation Assay, Knock-Out, Transgenic Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Aging‐Associated Nox4 ‐Mediated Mitochondrial Reactive Oxygen Species and DNA Damage Promote Vascular Cell Reprogramming and Aortic Remodeling in Abdominal Aneurysms

doi: 10.1161/JAHA.125.044949

Figure Lengend Snippet: A , B , Flow cytometry analysis of single‐cell suspension from the abdominal aorta of mice treated with Ang II. A , presents the quantification of MYH11 + cells; 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. These data show 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). ACTA2 indicates actin alpha 2; Ang II, angiotensin II; Apoe −/− , apolipoprotein E knockout mice; MYH11, myosin heavy chain 11; cGAS, cyclic GMP‐AMP synthase; CNN, Calponin 1; IL‐1b, interleukin 1 beta; IL‐6, interleukin 6; MYH11, myosin heavy chain 11; Nox4 TG, Nox4 transgenic mice; Nox4 −/− , Nox4 knockout mice; SMC, smooth muscle cell; STING, stimulator of interferon gene; TAGLN, transgelin; TNFα, tumor necrosis factor alpha; t‐SNE, t‐distributed stochastic neighbor embedding; and TUBB, tubulin beta.

Article Snippet: The immunostaining was conducted using the following antibodies: NOX4, TOMM20 (translocase of outer mitochondrial membrane 20)‐AlexaFluor488, IL1b (Abcam: ab109225, ab205486, ab254360); FGA, DNASE2 (deoxyribonuclease II; Thermo Fisher: PA5‐141128, PA5‐115136); 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), ATP5G (ATP synthase membrane subunit C locus1)‐AlexaFluor488, CD68‐AlexaFluor488, CD68‐Cy3, MYH11 (myosin heavy chain 11)‐AlexaFluor488 (Bioss, Woburn, MA: bs‐1278R, bs‐12547R‐A488, bs‐12547R‐A488, bs‐0649R‐Cy3, bs‐1956R‐A488); ACTA2‐FITC (actin alpha 2, smooth muscle), ACTA2‐Cy3 (Sigma‐Aldrich, St. Louis, MO: F3777, C6198); CD11b (Abnova, Walnut, CA: PAB12136 ); cyclic GMP‐AMP synthase (cGAS), STING (stimulator of interferon genes), and IL6 (interleukin‐6; Cell Signaling Technology, Danvers, MA: 15102S, 13647S, 12912S).

Techniques: Flow Cytometry, Single Cell, Suspension, Expressing, Fluorescence, Knock-Out, Transgenic Assay