Fig. 5

Fig. 5

The 4EHP and TRS interaction is critical for translation initiation of mRNAs required for vascular development. a Effects of TRS and/or 4EHP on VEGF secretion in siTRS- and/or si4EHP-transfected cell lines. VEGF protein levels in the culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA). b Effects of TRS and/or 4EHP on ANG secretion in siTRS- and/or si4EHP-transfected cell lines. ANG protein levels in the culture supernatant were determined by ELISA. c, d Effects of TRS and/or 4EHP on endothelial cell tube formation. Culture medium from WI-26 cells transiently transfected with siRNAs against TRS and/or 4EHP (or siCont) was used to treat HUVECs, which were subsequently plated on growth factor-reduced Matrigel to form capillary tubes (c). Total tube lengths in Supplementary Fig. 6a were measured using ImageJ (c). Migrated HUVECs were counted from three randomly selected fields (c). Culture medium from WI-26 cells transiently transfected with Myc-TRS WT or its 4EHP-binding-defective M60K mutant was used to treat HUVECs, which were subsequently plated on growth factor-reduced Matrigel to form capillary tubes (d). Total tube lengths in Supplementary Fig. 6f were measured using ImageJ (d). Migrated HUVECs were counted from three randomly selected fields (d). VEGF (10 ng mL^-1) served as a positive control. siCont, non-targeting control siRNA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. control group. Values are means ± SD of three independent experiments (a−d). e−h TRS- and 4EHP-mediated translation initiation is critical for vascular development. TRS was suppressed using a morpholino (trs i6e7 MO) together with control (Cont) RNA, or reconstituted with WT or I55D mutant trs RNA at 52 h post-fertilization (e). 4EHP was suppressed using a morpholino (4ehp MO) together with Cont RNA, or reconstituted with WT 4ehp RNA (g). Quantitation and statistical analysis of the lengths and branching points of central arteries in the hindbrain of Tg (kdrl:EGFP) zebrafish embryos (f, h). Scale bar = 100 μM. The number of analyzed zebrafish embryos is shown in parentheses. ***p < 0.001; NS not significant vs. control group. Values are means ± SD. SF serum-free medium