Fig. 4: Robo4 modulates Endo-MT through AKT/Smad signaling pathways.

A Pseudo tube formation assay. The graphs represent means ± SEM of the total tube length and branching point per field determined by quantifying at least 3 areas per well; Scale bars, 200 µm. B The migration ability of cells within 48 h was detected by scratch assay. Typical images were obtained at 0 h, 24 h, and 48 h using ×40 amplification, and the migration rate within 48 h was compared as means ± SEM; n ≥ 3. C Transwell assay indicates that Robo4 overexpression inhibits IR-induced migration of ECs. The analytical data are presented as mean ± SEM; n ≥ 3. D After the cells were transfected with Lentiviral shRobo4 and shControl in the presence or absence of ionizing radiation, the proteins in the PI3K/AKT/mTOR pathway of the ECs were evaluated using western blot. Data are the mean of three independent experiments, n ≥ 3. Error bars represent SEM, and data were analyzed by one-way ANOVA and Tukey’s post hoc test. E Immunoblots and quantification of phosphorylated p65 (NF-κB) and IκBα expressions in ECs with or without Robo4 depletion and IR. Data are shown as means ± SEM; n ≥ 3. F Protein expression levels of p-Smad1, Smad1, p-Smad2, and Smad2 in ECs treated with shRNA for Robo4 and ionizing radiation. Data are the mean of three independent experiments, and error bars represent SEM. Expression of Snail1 was detected by immunoblotting and Immunofluorescence staining in microvascular ECs lacking (G) or (H) overexpressing Robo4 treated with γ-radiation. Data are shown as means ± SEM; n ≥ 3. Scale bar, 20 µm; *ρ < 0.05; **ρ < 0.01; ***ρ < 0.001; ****ρ < 0.0001).