Fig. 1.

CCL9 is up-regulated in the myeloid-tumor co-culture supernatant and premetastatic lung. (A) Cytokine protein array, CCL9 and family members are highlighted with colored boxes. One of two experiments performed is shown here. Right upper panel: semi-quantitative data from dot density. (B-C) CCL9 ELISA of culture supernatant from myeloid-tumor co-culture, myeloid or tumor cell single culture, as well as protein extraction from pellet of 4T1 (B) or B16BL6 (C) tumor cells. For both (B) and (C), Gr-1+CD11b+ cells were pooled from 3 mice, with technical triplicates per treatment group. (D-E) CCL9 ELISA of premetastatic lung protein extraction from 4T1 tumor-bearing mice (D) and B16F10 tumor-bearing mice (E). n=3 mice for each group. Equal amount of proteins extracted from lungs were loaded to the plate and the results were presented as pg CCL9/ug protein. (F) CCL9 expression in myeloid cells cultured in 4T1 tumor-conditioned media or in co-culture with 4T1 tumor cells, ELISA assay with triplicates of samples. The experiment was repeated 2 times with different biological samples. (G) CCL9 ELISA of sorted Gr-1+CD11b+ cells and lungs from mice received intraperitoneally injection of hypoxic 4T1 conditioned medium for 14 days (n=5 mice). *P<0.05, **P<0.01, ***P<0.001.