rTMD1 suppresses EGF-mediated HUVEC migration and tube formation. (A) Pichia-expressed rTMD1 inhibits EGF-mediated HUVEC migration. EGF-containing media was added to the bottom compartment of a Boyden chamber as chemoattractant, and rTMD1 was preincubated with HUVECs 30 minutes before being added to the upper compartment. (B) Multivalent LeY Ag blocks the rTMD1-mediated inhibitory effect on EGF-elicited endothelial cell migration. The Boyden chamber assay was performed. rTMD1 was mixed with multivalent LeY (LeY-PAA) in cell solution, and EGF was used as chemoattractant. ***P < .001 compared with the control. #P < .05, ##P < .01, and ###P < .001 compared with EGF alone. The data are the mean ± SD (n = 6). Similar results were obtained in at least 3 independent experiments. (C-D) rTMD1 inhibits tube formation induced by EGF. (C) Schematic presentation of tube structure on Matrigel after 14 hours of incubation. (D) Statistical analysis of the tube length in each group. The magnification bar is 20 μm. The data are the mean ± SD (n = 3). Similar results were obtained in at least 3 independent experiments. **P < .01 and ***P < .001 compared with the control; ###P < .001 compared with EGF alone.