Fig. 4

CSF-1 and CCL2 contribute to cancer cell proliferation and activity. Macrophages (a (i)) and REM134 (a (ii)) cells were incubated with increasing doses of CSF-1. Cellular proliferation was measured after 48 h. PBS + 0.1 % BSA was used a vehicle control at 0 ng/ml. REM134 cells were incubated with increasing concentrations of CCL2 (a (iii)) or conditioned media (CM) from CCL2 treated macrophages (a (iv)). Cellular proliferation was measured after 48 h. PBS + 0.1 % BSA was used a vehicle control at 0 ng/ml. Experiments are replicates of 6. Statistical analysis considered each group against the control group. Expression of CSF-1R was assayed in Lilly cancer cells, after exposure to macrophage conditioned media, by immunofluorescence (b (i)). Expression of CCR2 was measured in REM134 cells, after exposure to macrophage-conditioned media, by qRT-PCR (b (ii)). Cellular activity was measured by glucose uptake. REM134 cells were pre-incubated with macrophage-conditioned media for 72 h prior to analysis. Ethanol was used as a vehicle control (VC). Cells were incubated at 4 °C as a negative control (c). Figures B and C are replicates of 3. Statistical analysis considered each group against the REM group. Asterisks or horizontal bars indicate statistically significant differences to the 0 ng/ml or REM group, where P < 0.05 by Kruskal-Wallis (Figure A) or Mann–Whitney (Figures B and C); “ns” indicates non-significant differences