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. 2015 Mar 27;30(6):1290–1301. doi: 10.1093/humrep/dev067

Figure 4.

Figure 4

E2-dependent secretion of CCL2 regulates uNK-mediated angiogenesis. HEEC network formation was assessed in response to recombinant human CCL2 (rCCL2). (A) Network formation was significantly increased in response to 0.1 ng/ml rCCL2 (n = 4, One-sample t-test, P = 0.0348) and maximal in response to 0.5 ng/ml (n = 4, One-sample t-test, P = 0.047). (B) HEEC were incubated with 0.5 ng/ml rCCL2 and increasing concentrations of a monoclonal antibody to CCL2 (mAb-CCL2). HEEC network formation was assessed relative to 0 µg/ml mAb-CCL2. Neutralization of rCCL2 with mAb-CCL2 significantly inhibited HEEC network formation at concentrations of 0.1 µg/ml (P = 0.0174) and greater (n = 4, One-sample t-test, 0.5 µg/ml; P = 0.0026, 1 and 5 µg/ml; P = 0.0005). (C) uNK-E2 CM was incubated with 0.5 µg/ml mAb-CCL2 and HEEC network formation was assessed relative to control uNK CM (DMSO). Neutralization of CCL2 significantly reduced HEEC network formation (n = 8, One-sample t-test, P = 0.0069) and reversed uNK-E2-mediated increased network formation (n = 8, Mann–Whitney, P = 0.0006). *P < 0.05, **P < 0.01, ***P < 0.001. Images were captured at ×5 magnification using the Axiovert 200 Inverted Fluorescent Microscope.