Fig. 5 | Nature Communications

Fig. 5

From: miR-103 promotes endothelial maladaptation by targeting lncWDR59

Fig. 5

Role of lncWDR59 on hyperlipidemia-mediated DNA damage and MN formation in ECs. a Immunofluorescence analysis of Ki67 or phosphorylated gamma H2AX histone (γH2AX) in MAoECs treated for 24 h with 25 or 100 µg ml−1 oxLDL. Data are represented as percentage of total number of cells (n = 4 per group). b The en face 3D reconstructed arch and thoracic aortae from Apoe−/− mice fed 12 weeks of ND or HFD and stained for γH2AX and CD31. The graph represents the number of CD31+ cells with a positive γH2AX staining in the nucleus, normalized on total number of CD31+ cells and expressed in percentage (n = 4 mice per group). c Analysis of nuclear γH2AX+ vWF+ in the root of EC-Dicerflox mice compared to EC-DicerWT mice, fed 12 weeks of HFD, and expressed as percentage of total vWF+ cells (n = 3 mice per group). d Analysis of nuclear γH2AX+ MAoECs treated with TSBs for 24 h, and stimulated with or without 25 µg ml−1 oxLDL. Data are normalized on total number of ECs and expressed in percentage (n = 4–6 per group). e MAoECs were treated for 24 h with DAPT or siCtnnb1 to analyze the nuclear γH2AX+ staining. The same treatments were performed in 25 µg ml−1 oxLDL-treated MAoECs, alone or in combination with TSB, to analyze the nuclear γH2AX+ staining. Data are normalized on total number of cells and expressed in percentage (n = 3–5 per group). f Analysis of micronucleated CD31+ cells (MN cells) and CD31+ cells with γH2AX+ micronuclei (γH2AX+ MN) on en face 3D reconstructed arch and thoracic aortae from Apoe−/− mice fed 12 weeks of ND or HFD (n = 3 mice per group). g Analysis of MN formation and γH2AX+ MN from micronucleated MAoECs (γH2AX+ MN) treated for 24 h with 25 or 100 µg ml−1 oxLDL (n = 3 per group). Micronucleated MAoECs treated or not with 25 µg ml−1 oxLDL, captured using confocal microscope. Transversal section shows γH2AX staining around the heterochromatin of principal nucleus and inside of the MN. h Analysis of MN formation and γH2AX+ MN in micronulceated MAoECs treated with TSBs for 24 h, and stimulated with or without 25 µg ml−1 oxLDL. Data are normalized on total number of ECs and expressed in percentage (n = 4–10 per group). i Analysis of MN formation and γH2AX+ MN MAoECs treated for 24 h with DAPT or siCtnnb1, in combination with 25 µg ml−1 oxLDL (n = 3–10 per group). DMSO dimethyl sulfoxide, DAPT γ-secretase inhibitor. Data are represented as mean ± SEM of the indicated number (n) of repeats. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t-test. #P < 0.05 versus all other groups by one-way ANOVA and two-way ANOVA. Scale bar: 25 µm

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