Figure 3: SMAD6 is Notch regulated and anti-angiogenic.
From: Notch regulates BMP responsiveness and lateral branching in vessel networks via SMAD6
(a) qRT–PCR for HES1 or SMAD6 with Notch gain-of-function (Dll4-Fc) or loss-of-function (DAPT). Data points, independent experiments (n=5 DLL4-Fc and 9 DAPT), log-conversion of the DDCT versus controls. Quantification (b) and panels (c) of nuclear SMAD6 expression in individual HUVEC with indicated parameters, representative of two independent experiments. Scale bar, 10 μm. Yellow arrow, EC expressing FLAG-NICD. Error bars, mean±s.e.m., ****P<0.0001 by one-way analysis of variance using Tukey’s post-hoc; NS, not significant. (d) Stable lentivirally transduced HUVEC infected with SMAD6-tdTomato conditonally expressing virus, stimulated with doxycycline (DOX) and stained as indicated. (e) Quantification of twofold dose–response (indicated on x axis) to DOX (SMAD6-tdTomato expression) after 90 min BMP6 treatment, representative of two independent experiments. Readout is nuclear pSMAD1/5. Data are four-parameter best-fit curves (solid lines) ±95% confidence bands (filled areas). (f) HUVEC transfected with indicated siRNAs, then treated with 40 ng ml−1 BMP6 for 90 min before fixation and staining for nuclear pSMAD1/5, representative of two independent experiments (n=75 NT and 80 SMAD6 siRNA HUVEC). Error bars, mean±95% CI. ****P≤0.0001 by Student’s t-test. (g–j) HUVEC 3D sprouting assay with indicated siRNAs and treatment, visualized with phalloidin (actin) and depth-encoded in a compressed z-stack, representative of three independent experiments. (k) Quantification of branching (n=11 NT/control, 13 NT/BMP6, 12 SMAD6 siRNA/control and 11 SMAD6 siRNA/BMP6 beads). Data points, individual beads; bars, mean±95% CI. **P≤0.01 and ***P≤0.001 by Kruskal–Wallis with Dunn’s post-hoc test.
