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(A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by <t>CD144</t> (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.
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(A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by <t>CD144</t> (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.
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(A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by <t>CD144</t> (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.
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(A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by <t>CD144</t> (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.
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Image Search Results


(A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by CD144 (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.

Journal: bioRxiv

Article Title: DOT1L-AF10–mediated H3K79me3 promotes NF-κB p65–dependent inflammatory activation in endothelial cells

doi: 10.64898/2026.03.20.713137

Figure Lengend Snippet: (A-B) Immunofluorescence staining of histological sections from partially ligated (D-Flow) and contralateral unligated (S-Flow control) carotid arteries of HFD-fed C57BL/6 mice (n = 3), performed four weeks post-surgery, showing endothelial expression of DOT1L (A) and H3K79me3 (B). EC are marked by CD144 (green), nuclei by DAPI (blue), and target signals (DOT1L or H3K79me3) in red. Fluorescence intensities (AU) in individual EC (dots) from three biological replicates are indicated together with the mean. A total of ≥ 45 EC were analyzed per condition. (C-E) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-DOT1L antibody (red, C) or H3K79me3 antibody (red, D, E) in both lesser (disturbed flow [D-flow]) and greater (steady laminar flow [S-flow]) curvature areas including TNF-α treated aortic tissue (E). Images were obtained from the luminal surface of the aorta. The levels of nuclear EZH2 and H3K27me3 were analyzed using Image J software. (n = 3) Magnification: x10, x63; Scale: 200 µm, 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Welch’s t-test.

Article Snippet: For tissue sections, permeabilization was performed using 0.5% Triton X-100, followed by blocking with 10% goat serum supplemented with anti-mouse IgG (1:1000) for 1 h. Cells and tissue sections were incubated overnight at 4°C with the following primary antibodies: DOT1L Rabbit mAb (1:500; #90878), H3K79me3 Rabbit mAb (1:500; #74073), eNOS Rabbit mAb (1:100; #32027), ICAM1 Rabbit pAb (1:500; #4915), NF-κB p65 Rabbit mAb (1:800; #8242, Cell Signaling Technology, Danvers, USA), H3K79me3 Rabbit pAb (1:50; #PA5-96121), AF10 Rabbit pAb (1:500; #BS-3696R), AF17 Rabbit pAb (1:1000; #A302-198A, Invitrogen), and CD144 Mouse mAb (1:25; #SC-9989, Santa Cruz Biotechnology, Dallas, USA).

Techniques: Immunofluorescence, Staining, Control, Expressing, Fluorescence, Marker, Software