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. 2011 Aug 16;30(20):4142–4156. doi: 10.1038/emboj.2011.298

Figure 6.

Figure 6

Cortactin is crucial for angiogenesis and mediates HDAC6-dependent sprouting. (A) HUVECs were transfected with cortactin or control siRNA for 48 h. Cortactin expression was measured by western blot. Expression of HDAC6 and α-tubulin were used as loading control (n=3). (B) HUVECs were transfected with cortactin or control siRNA for 48 h, followed by a transwell migration assay for 4 h under basal (black bars) or stimulatory conditions with 50 ng/ml VEGF (grey bars). Migrated cells were counted manually (n=4). (C) HUVECs were transfected with cortactin or control siRNA for 24 h, followed by a spheroid assay (n=3). (D) Representative images of tg(fli1:EGFP) zebrafish embryos injected with cortactin splice-blocking Mo (cortactin-SB-Mo) at 48 h post fertilization. Upper panel shows transmitted light images of the whole embryo. Lower panel shows fluorescent confocal images of the anterior part. White arrows indicate vessel defects, grey arrows indicate faint connections of the DLAV and red arrows indicate the absence of the parachordal vessel. (E) HUVECs were transduced with cortactin wt and different mutated cortactin constructs for 24 h, followed by a spheroid assay. For deacetylation mimic cortactin (cortactin 9KR), nine lysine residues of the repeat region were mutated to arginines. For acetylation mimic cortactin (cortactin 9KQ), nine lysine residues of the repeat region were mutated to glutamines. Mock-transduced cells serve as control (n=5). (F) HUVECs were stably transduced with HDAC6 wt virus or control virus, and subsequently transfected with either cortactin or control siRNA for 24 h. Spheroid assay was performed, and the cumulative sprout length was measured (n=4). (G) HUVECs were stably transduced with HDAC6 or control shRNA virus, and subsequently transduced with mock control or different cortactin constructs (see figure legend E). (H) Quantification of cumulative sprout length in a spheroid assay (n=5). (H) Quantification of ISV and DLAV vessel defects in ATG-HDAC6 morphants with or without 100 pg of cortactin 9KR-mRNA 48 h post fertilization. As control group serves 100 pg of cortactin 9KR-mRNA. Data are presented as percentage (%) of ATG-HDAC6-Mo. *P<0.05 for ISV; #P<0.05 for DLAV (n=56 for ATG-HDAC6-Mo; n=49 for ATG-HDAC6-Mo+cortactin 9KR-mRNA; and n=48 for cortactin 9KR-mRNA). Figure source data can be found with the Supplementary Information.