Fig. 3 | Nature Communications

Fig. 3

From: miR-103 promotes endothelial maladaptation by targeting lncWDR59

Fig. 3

miR-103 target lncWDR59 regulates EC proliferation through Wnt and Notch1 signaling pathways. MAoECs were transfected for 24 h with miR-103 inhibitors (miR-103 inh), lncWDR59 Gapmers (GlncWDR59), or TSB (a) to check Ccl2 and Cxcl1 relative expression (n = 3–6 per group), or (b) to stain with an anti-Ki67 antibody to analyze their proliferation and cdkn1a expression by qPCR (n = 4–8 per group). DAPI was used for nuclei staining. EC proliferation was calculated as number of Ki67+ cells on total number of cells and expressed in percentage (n = 4 per group). c MAoECs were seeded in ibidi flow chamber slides and incubated with EdU and TSB or control LNAs. ECs were subjected for 48 h to low (LSS; 5.51 dyn cm−2) or high (HSS; 10 dyn cm−2) shear stresses and stained for EdU-DNA incorporation (n = 3–4 per group). d MAoECs were treated with DAPT or siRNAs against β-catenin (siCtnnb1) for 24 h and stained with anti-Ki67 antibody to analyze their proliferation as described before. DMSO and siRNA were used as controls of DAPT and siCtnnb1, respectively (n = 4–8 per group). e, f MAoECs were transfected with miR-103 inhibitors, TSBs, or GlncWDR59 as described before and stained with an anti-activated Notch1 (NICD) (e) or β-catenin antibody (f). Data were represented as number of nuclear NICD+, and nuclear (star) and perinuclear (arrowheads) β-cat+ localization. Inhibition of β-catenin activation makes β-catenin visible only as membrane adaptor protein for cell-to-cell intercellular adhesions. Data were normalized on total number of cells and expressed in percentage (n = 4–8 per group). gj MAoECs were treated for 24 h with DAPT or siCtnnb1, alone or in combination with TSB to analyze (g) EC proliferation by Ki67 staining. DMSO or siRNAs in combination with LNA-controls were used as controls. Data were normalized on total number of cells and expressed in percentage (n = 4 per group). h Analysis of Ki67+ TSB+siCtnnb-transfected MAoECs stimulated with DAPT or DMSO for 24 h (n = 4 per group). Analysis of β-catenin (i) or NICD (j) nuclear localization. The graphs correspond to the analysis of nuclear (stars) and perinuclear (arrowheads) β-cat+ and nuclear NICD+ localization. Data were normalized on total number of cells and expressed in percentage (n = 4–8 per group). Ccl2 C-C motif chemokine ligand 2, Cxcl1 C-X-C motif chemokine ligand 1, cdkn1a cyclin-dependent kinase inhibitor 1a, TSB target site blockers, 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 one-way ANOVA and t-test. Scale bar: 25 µm

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