Fig. 6

Fig. 6

FOXO1 activation is required for VEGFA-induced regression in zebrafish cds2 mutants. a Western blotting analysis shows that Foxo1a translocates to the nuclei in cds2-deficient zebrafish endothelium upon VEGFA stimulation. Protein samples for whole embryos or cytosol/nucleus fractions were collected at 48 hpf from Tg(fli1a:gal4) embryos with Tol2 transposase-mediated Tg(uas:myc-foxo1a) transgenesis, with or w/o cds2 MO and with or w/o vegfa OE (heat-shock induction at 28 hpf) as indicated in the figure. b Western blotting analysis shows that pten knockdown blocks Foxo1a nuclear accumulation in cds2-deficient zebrafish endothelium with vegfa OE. Protein samples for total/cytosol/nucleus fractions were collected at 48 hpf from cds2 MO-injected Tg(fli1a:gal4) embryos with Tol2 transposase-mediated Tg(uas:myc-foxo1a) transgenesis and vegfa OE, with or w/o pten MO. Representative images (c) and phenotype quantitative analysis (d) of trunk vessels in cds2 mutant embryos with or w/o vegfa OE (heat-shock induction at 28 hpf) and with or w/o AS1842856 treatment (from 24 hpf) at 76–80 hpf. AS, AS1842856. The ISV number quantified on the top (d) is from 20–24 embryos per group. e Relative vegfa mRNA level in cds2 mutants with heatshock-induced vegfa OE, treated with DMSO or AS. The vegfa expression level was determined at 2 h post heat shock induction and normalized to WT embryos without vegfa OE. Ctrl, DMSO; AS, AS1842856. n = 3 samples, 20 embryos pooled for each sample. Scale bar, 100 μm. Error bar, mean ± SEM. ns, not significant (P ≥ 0.05)