Fig. 4

qRT-PCR analysis of CD163, CD206, CCL1, CCL22, IL-12, CD86, iNOS and HLA in M-DM stimulated for 24 h with CM from Bcl-xL overexpressing A) Mxl90 and B) Mxl7 melanoma cells with or without anti-IL-1β (ABIL-1β) or anti-IL-8 (ABIL-8) blocking antibodies. Percentage of mRNA variation relative to untreated cells is reported. p-values were calculated between Bcl-xL overexpressing cells untreated and treated with antibody. C) Representative images (left panels) and relative quantification (right panel) of M-DM migration in response to CM from control (CM Mneo), Mxl90 untreated (CM Mxl90) or treated with the blocking antibody CCL5 (CM Mxl90 + ABCCL5). The quantification was performed by counting the number of migrated cells in at least 10 fields for each condition. p-values were calculated between M-DM migrated cells exposed to CM derived from Bcl-xL overexpressing clones treated and untreated with antibody. D) Representative images of Mneo, Mxl90 and Mxl90 cells silenced for CCL5 (Mxl90 siCCL5) stained in green 5 days after injection in the yolk sac of 2 days post fertilization Tg(mfap4:Tomato) zebrafish larvae, and relative quantification of E) recruited macrophages, F) interaction between melanoma cells and macrophages counted as yellow spots (green arrows), and G) invasion score of melanoma cells. p-values were calculated between E, G) number of melanoma cells and macrophages recruited in the tail of zebrafish larvae silenced or not for CCL5, F) number of interaction between macrophages and melanoma cells silenced or not for CCL5. *p < 0.05, **p < 0.01. ***p < 0.001