Figure 5
Figure 5. Slit3 interacts with Robo4 but not Robo1 to induce EC chemotaxis and Rho GTPases activation and triggers intracellular actin cytoskeleton reorganization. (A) Anti-Robo4 but not anti-Robo1 antibody blocked Slit3-induced EC migration. Nonimmune antibody (Ni-Ab, 1 μg), functional blocking anti-Robo1 (R1-Ab, 1 μg), or functional blocking anti-Robo4 (R4-Ab, 1 μg) antibody was supplemented in the bottom chamber containing BSA (1.3 nmol/L), Slit3 (0.8 nmol/L), or VEGF (1.5 nmol/L). Slit3-induced HUVEC migration was quantified. The experiments were carried out at least 3 times in triplicate. The data are presented as mean ± SD. ** indicates P < .001 when a comparison was made with the wells treated with Slit3 and Ni-Ab by Student t test. (B). Soluble extracellular domain of Robo4 (sR4) but not the soluble extracellular domain of Robo1 (sR1) inhibited Slit3-induced HUVEC migration. Slit3- and VEGF-induced HUVEC migration was similarly carried out as described in panel A with supplement of heat-inactivated sR1, heat-inactivated sR4, active sR1, or active sR4 (each at 1 μg) in the lower chamber. Only the active sR4 showed an inhibitory effect on the Slit3-induced HUVEC migration. Furthermore, neither Robo1 nor Robo4 affected the VEGF-directed EC migration. (C). CM collected from HUVEC and VSMC culture induced Robo4-dependent HUVEC migration. The CM was collected from serum-free culture of HUVECs (EC-CM) and VSMCs (VSMC-CM), respectively, and tested for their promigratory activity as described in panel A with or without the addition (1 μg) of Ni-Ab or R4-Ab. The R4-Ab but not the Ni-Ab partially inhibited the CM-induced HUVEC migration, suggesting that endogenous Slit3 interacts with Robo4 to induce EC migration to promote angiogenesis. (D) Slit3 activates Rho GTPases. HUVECs before and after Slit3 stimulation were lysed, and the GST pull-down assays were carried out to quantitate the GTP binding form of endogenous Rho GTPases (the active form, a-). The total Rho GTPases (t-) were detected with corresponding antibody by Western blot analysis. The bands were quantified by density measurement. All the experiments were performed 3 times independently. (E). Anti-Robo4 antibody blocked Slit3-induced activation of Rho GTPases. Ni-Ab (1 μg) or R4-Ab (1 μg) was supplemented in culture medium before HUVECs received Slit3 (0.8 nmol/L) stimulation. After 15 minutes of the Slit3 treatment, the HUVECs were lysed for Rho GTPase analyses. (F) Slit3 triggers actin cytoskeleton reorganization in ECs and formation of stress fiber and lamelipodia. Subconfluent HUVECs were serum-starved overnight, treated with Slit3 (0.8 nmol/L) or BSA (1.3 nmol/L) for 30 minutes, and then stained with TRITC-phalloidin. Fluorescence images were captured at ×20 magnification. Arrowheads indicate actin stress fibers (white) and lamellipodia (green). Scale bar, 20 μm.

Slit3 interacts with Robo4 but not Robo1 to induce EC chemotaxis and Rho GTPases activation and triggers intracellular actin cytoskeleton reorganization. (A) Anti-Robo4 but not anti-Robo1 antibody blocked Slit3-induced EC migration. Nonimmune antibody (Ni-Ab, 1 μg), functional blocking anti-Robo1 (R1-Ab, 1 μg), or functional blocking anti-Robo4 (R4-Ab, 1 μg) antibody was supplemented in the bottom chamber containing BSA (1.3 nmol/L), Slit3 (0.8 nmol/L), or VEGF (1.5 nmol/L). Slit3-induced HUVEC migration was quantified. The experiments were carried out at least 3 times in triplicate. The data are presented as mean ± SD. ** indicates P < .001 when a comparison was made with the wells treated with Slit3 and Ni-Ab by Student t test. (B). Soluble extracellular domain of Robo4 (sR4) but not the soluble extracellular domain of Robo1 (sR1) inhibited Slit3-induced HUVEC migration. Slit3- and VEGF-induced HUVEC migration was similarly carried out as described in panel A with supplement of heat-inactivated sR1, heat-inactivated sR4, active sR1, or active sR4 (each at 1 μg) in the lower chamber. Only the active sR4 showed an inhibitory effect on the Slit3-induced HUVEC migration. Furthermore, neither Robo1 nor Robo4 affected the VEGF-directed EC migration. (C). CM collected from HUVEC and VSMC culture induced Robo4-dependent HUVEC migration. The CM was collected from serum-free culture of HUVECs (EC-CM) and VSMCs (VSMC-CM), respectively, and tested for their promigratory activity as described in panel A with or without the addition (1 μg) of Ni-Ab or R4-Ab. The R4-Ab but not the Ni-Ab partially inhibited the CM-induced HUVEC migration, suggesting that endogenous Slit3 interacts with Robo4 to induce EC migration to promote angiogenesis. (D) Slit3 activates Rho GTPases. HUVECs before and after Slit3 stimulation were lysed, and the GST pull-down assays were carried out to quantitate the GTP binding form of endogenous Rho GTPases (the active form, a-). The total Rho GTPases (t-) were detected with corresponding antibody by Western blot analysis. The bands were quantified by density measurement. All the experiments were performed 3 times independently. (E). Anti-Robo4 antibody blocked Slit3-induced activation of Rho GTPases. Ni-Ab (1 μg) or R4-Ab (1 μg) was supplemented in culture medium before HUVECs received Slit3 (0.8 nmol/L) stimulation. After 15 minutes of the Slit3 treatment, the HUVECs were lysed for Rho GTPase analyses. (F) Slit3 triggers actin cytoskeleton reorganization in ECs and formation of stress fiber and lamelipodia. Subconfluent HUVECs were serum-starved overnight, treated with Slit3 (0.8 nmol/L) or BSA (1.3 nmol/L) for 30 minutes, and then stained with TRITC-phalloidin. Fluorescence images were captured at ×20 magnification. Arrowheads indicate actin stress fibers (white) and lamellipodia (green). Scale bar, 20 μm.

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