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. 2017 Jul 21;31(11):4720–4733. doi: 10.1096/fj.201700193RR

Figure 5.

Figure 5.

BMP-induced endothelial sheet migration is independent of VEGF and requires p38 and HSP27. A) Representative images of HUVEC sheets treated with 10 nM BMP2, 10 nM BMP6, or 2 nM VEGF for 16 h. Dashed lines indicate endothelial sheet borders. Scale bars, 200 µm. B) Quantification of HUVEC sheet migration. Means ± sd; n = 8. ***P < 0.001 vs. control. C) Quantification of HUVEC sheet migration in the presence of 1 µM K02288. Means ± sd; n = 3. ***P < 0.001 vs. control; ###P < 0.001 vs. VEGF-treated control without K02288 (as quantified in B). D) Quantification of HUVEC sheet migration in the presence of 2 µg/ml human VEGF neutralizing antibody. Means ± sd; n = 5. ***P < 0.001 vs. control; ###P < 0.001 vs. VEGF-treated control without anti-VEGF Ab (as quantified in B). E) Quantification of BMP-induced HUVEC sheet migration in the presence of 20 µM SB203580. Means ± sd; n = 8. ###P < 0.001 vs. BMP-treated control without SB203580 (as quantified in B). F) Immunoblot analysis of indicated proteins in HUVECs upon BMP2 or BMP6 stimulation for 45 min in the absence or presence of 20 µM SB203580. G) Quantification of BMP-induced sheet migration in HUVECs transfected with siRNA targeting either HSPB1 or control sequences (scrambled; si-scr). Means ± sd; n = 4. ***P < 0.001 vs. control; ###P < 0.001 vs. BMP-treated si-scr control. H) Representative images of HUVEC spheroids transfected with siRNAs targeting HSPB1 or control sequences (scrambled; scr). Dashed lines highlight spheroid sprouting area. Scale bars, 100 µm. I) Quantification of total sprout length. Means ± sd; n = 12 from 4 independent experiments. Control values are the same as depicted in Fig. 3B, as experiments were run in parallel. ***P < 0.001. J) Representative confocal images of HUVEC spheroids transfected with indicated siRNA and stained with phalloidin (actin; green) and DAPI (DNA; blue). Scale bars, 50 µm. K) Quantification of tip cell nuclei per spheroid. Means ± sd; n = 12 from 4 independent experiments. Control values are the same as depicted in Fig. 3D, as experiments were run in parallel. ***P < 0.001.