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Figure 2
(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs in 3D angiogenic sprouting assay. Sprouts were stained for Phalloidin (actin) and then depth encoded such that cooler colors are further in the Z-plane and warmer colors are closer in the Z-plane. Scale bar, 100 µm. (B) Quantification of average sprout length of 3D angiogenic sprouts shown in (A). n=42 beads (non-targeting [NT]) and 43 beads (SUN1 knockdown [KD]) compiled from five replicates. ****, p<0.0001 by Student?s two-tailed unpaired t-test. (C) Quantification of branches/mm of 3D angiogenic sprouts shown in (A). n=41 beads (NT) and 43 beads (SUN1 KD) compiled from five replicates. ****, p<0.0001 by Student?s two-tailed unpaired t-test. (D) Stills from Video 1 and Video 2 showing sprouting dynamics of HUVEC with indicated siRNAs over 50 hr. Scale bar, 50 µm. (E) Quantification of HUVEC sprout extensions and retractions shown in (D). n=101 sprouts (NT) and 77 sprouts (SUN1 KD) compiled from three replicates. p<0.001 by ?2 analysis. (F) Representative images of HUVEC with indicated siRNAs and stained with indicated antibodies in the 3D sprouting angiogenesis assay. Endothelial cells were stained for DAPI (cyan, DNA), Phalloidin (green, actin), and VE-cadherin (white, junctions). Arrows indicate normal junctions; arrowheads indicate abnormal junctions. Scale bar, 20 µm. (G) Quantification of disorganized VE-cadherin as shown in (F). n=32 junctions (NT) and 30 junctions (SUN1 KD) compiled from two replicates. ****, p<0.0001 by Student?s two-tailed unpaired t-test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.
Nuclear SUN1 is required for sprouting angiogenesis.