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-E) Immunoblot analyses of EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of the DOT1L inhibitor SYC-522 (5 µM, 24 h), showing the expression of H3K79me3 (n = 4; A), eNOS (n = 5; B), ICAM1 (n = 3; C), Phospho-NF-κB p65 (n = 4), total NF-κB p65 (n = 3), Phospho-IκBα (n = 3), total IκBα (n = 3; D), Phospho-IKKα/β (n = 3), and total IKKβ (n = 3; E). (F) Analyzing the transcript levels of NF-κB p65 in HUVEC EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of DOT1L siRNA (siDOT1L, 40 nM). (n = 3) (G-I) Immunoblot analyses of rat aortic rings treated with TNF-α (10 ng/ml, 24 h) ex vivo in the presence or absence of the DOT1L inhibitor SYC-522 (5 µM, 24 h), showing the expression of eNOS (n = 3; G), VCAM1 (n = 3; H), and total NF-κB p65 (I, n = 3). (J) En face preparations were double stained with anti–VE-cadherin (green, EC marker) and an anti-NF-kB p65 antibody (red, C) in TNF-α and/or DOT1L inhibitor (SYC-522) treated aortic tissue. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s post hoc 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: Western Blot, Expressing, Ex Vivo, Staining, Marker

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-E) Immunoblot analyses of EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of DOT1L siRNA (siDOT1L, 40 nM), showing the expression of DOT1L (n = 4; A), H3K79me3 (n = 3; B), eNOS (n = 4; C), ICAM1 (n = 3; D), and NF-κB p65 (n = 3; E). (F) Immunofluorescence staining of EA.hy926 treated with TNF-α (10 ng/ml, 24 h) in the presence or absence of GFP-FBXL10 plasmid (pFBXL10, 250 ng/ml), showing NF-κB p65 expression (red). Nuclei are stained with DAPI (blue). Fluorescence intensities (AU) in individual EC (dots) from three independent experiments are indicated together with the mean. A total of ≥ 55 EC were analyzed per condition. Magnification: x63; Scale: 20 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc 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: Western Blot, Expressing, Immunofluorescence, Staining, Plasmid Preparation, Fluorescence

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-C) Immunofluorescence staining of EA.hy926 exposed to D-Flow (4 h; n = 3), showing the expression of H3K79me3 (A), eNOS (B), and NF-κB p65 (C). Nuclei are stained with DAPI (blue), and target signals corresponding to each respective protein are in red. Fluorescence intensities (AU) in individual EC (dots) from three independent experiments are indicated together with the mean. A total of ≥ 60 EC were analyzed per condition. Magnification: x20; Scale: 100 µm. (D) Monocyte adhesion assay: DiI-labeled THP-1 were incubated (30 min) with flow-exposed EA.hy926 (4 h; n = 4) in the presence or absence of SYC-522 (5 µM). Adherent monocytes (red dots) were counted in regions exposed to S-Flow and D-Flow. Magnification: x10; Scale: 100 µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc 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, Expressing, Fluorescence, Cell Adhesion Assay, Labeling, Incubation