Figure 5.
Generation of CCR5-stimulatory activities from HCC-1 [1–74] by conditioned media from human tumor cell lines. CHO-K1 cells coexpressing CCR5 and apoaequorin were used to test the biological activity generated from HCC-1[1–74] in conditioned media. (a) The medium from monolayer cultures of the cell lines DU-145, 143B, MCF-7, and MeWo, incubated without (gray bars) or after addition of 1 μM HCC-1[1–74] (white bars), was tested for CCR5-stimulatory activities. (b) Conditioned medium from the DU-145 cell line (gray bar) was incubated with 200 nM HCC-1[1–74] alone (black bar) or in the presence of various protease inhibitors: aprotinin (0.3 μM), leupeptin (1 μM), AEBSF (4 mM), soybean trypsin inhibitor (5 μM), α2-macroglobulin (20 nM), 1,10-phenanthroline (10 μM), EDTA (1 mM), pepstatin (1 μM), and E-64 (N-[N-[l-3-trans-carboxirane-2-carbonyl]-l-leucyl]-agmatine, 2.5 μM). The results in RLU represent the mean and SEM of triplicate data points and are representative of two independent experiments. Cont. Med., conditioned medium without added HCC-1[1–74].