Met is required for Sema4D-induced angiogenic activity. (A) HUVECs were transfected with siRNAs specific for Met, and the level of the Met protein present in total cellular extracts was evaluated. The figure shows a reduction of Met expression in transfected cells. (B) HUVECs were infected with lentiviruses encoding a MetGFP chimera, previously shown to act in a dominant-negative manner. Cell extracts were immunoprecipitated with antibodies directed against the extracellular portion of Met (α-extracellular Met), and Western blot was probed with either α-GFP (top blot) or α-extracellular Met (bottom blot) antibodies. The figure shows that MetGFP is expressed in infected cells at a level higher than endogenous Met. (C) Migration assay of control HUVECs, HUVECs transfected with Met-specific siRNAs, and HUVECs expressing the dominant-negative MetGFP. Controls varied according to experimental conditions: HUVECs transfected with siRNAs against GFP were used in the experiments performed on cells treated with Met-specific siRNAs; HUVECs infected with a lentivirus containing the GFP sequence were used in experiments performed with HUVECs-MetGFP. As shown, an impairment of cell migration in response to Sema4D was observed in cells displaying a decreased expression of Met because of siRNA treatment and in cells expressing MetGFP. No reduction of mitogenic ability was observed in these cells in response to VEGF-A₁₆₅. (D) Quantification of the sprouting assay shown in panel E. Data in panels C and D indicate means ± SD. (E) A reduction of Sema4D-induced sprouting was observed in cells displaying decreased expression of Met because of siRNA treatment and in cells expressing MetGFP. Spheroids of cells expressing MetGFP were observed under ultraviolet light: sprouts formed by cells in response to VEGF-A₁₆₅ appeared green (xii), indicating that the expression of MetGFP did not interfere with the response to VEGF-A₁₆₅. The few sprouts observed in response to Sema4D were not fluorescent (x, white arrow) and were thus thought to have originated from cells that did not express the dominant-negative MetGFP. Spheroids were also stained with a vital red-emitting fluorochrome to allow detection of all sprouts (xiii-xv). Images were captured with a 20×/0.70 air objective lens.