Figure 5. Biological effect of GTPCH-induced Ang-1 secretion on tumor Tie2 phosphorylation.

(A). Cell cultures were done and treated as for Figure 4. The culture medium was concentrated and measured for Ang-1 by ELISA. (B). 1 × 10⁴ of MDA-MB231 or BT474 cells/well was seeded in a 96-well plate. After incubation with the medium of the GCHtet-off or Tet-off-EV ± DMSO vehicle control, Dox (1μg/ml), or DAHP (5 mM) for 20 min, cells were fixed and treated as for Fig. 3. pTie2 was quantified using cell based ELISA assay. (C). Cell monolayers of HUVEC, MDA-MB231 or BT474 were incubated with the medium of Ang-1siRNA knockdown or SCR control in GCHtet-off or pervanadate positive control. After 20 min stimulation, pTie2 was quantified using cell based ELISA assay. All data are shown as mean ± SEM (*p <0.05 vs. DMSO control, Dox or DAHP, or siRNA knockdown vs SCRsiRNA, n = 3). (D). Schematic representation of a working model of the molecular mechanism by which formation of GTPCH/Ang-1 complex promotes tumour development.