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. 2014 Jul 21;8(8):1729–1746. doi: 10.1016/j.molonc.2014.07.009

Figure 4.

Figure 4

Conditioned media from senescent fibroblasts increase in vitro angiogenesis. A) EPC cells, after 24 h of serum starvation, were seeded into the upper compartment of a Boyden chamber. Cells were allowed to migrate through the filter toward the lower compartment containing CM of pre‐senescent or senescent fibroblasts medium. Cell migration was evaluated after Diff‐Quick staining by counting cells in six randomly chosen fields. The results are representative of three experiments with similar results. *p < 0.005 CM senescent cells vs CM pre‐senescent cells. B) HUVECs were seeded on Matrigel coated multiwell in the presence of CM of pre‐senescent or senescent cells for 6 h at 37 °C. Cord formation was examined by phase‐contrast microscopy. The total number of junctions for optical field is counted. Results are representative of three independent experiments. *p < 0.005 CM senescent cells vs CM pre‐senescent cells. C) HUVEC cells, after 24 h of starvation in 1% serum were seeded into the upper compartment of Boyden chamber. Cells were allowed to migrate through the Matrigel‐coated filter toward the lower compartment filled with CM of pre‐senescent or senescent fibroblasts. Cell invasion was evaluated after Diff‐Quick staining by counting cells in six randomly chosen fields. The results are representative of five experiments with similar results. *p < 0.005 CM senescent cells vs CM pre‐senescent cells. D) 2 × 103 HUVECs were seeded in triplicate in 96‐multiwell plates and serum starved for 24 h. Cells were grown for 48 h at 37 °C in the presence of CM of pre‐senescent or senescent fibroblasts. Cell proliferation was evaluated after Crystal Violet staining. The results are representative of three experiments with similar results. *p < 0.005 CM senescent cells vs CM pre‐senescent cells.