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. 2014 May 5;111(20):E2120–E2129. doi: 10.1073/pnas.1406655111

Fig. 9.

Fig. 9.

Coculture- and hypoxia-induced CCL5 and CSF1 expression is required for migration of bone marrow-derived macrophages (BM-Mϕ). (A) Bone marrow cells were isolated from the BALB/c mice and differentiated into macrophages (BM-Mϕ) by supplementing the culture media with CSF1. The efficiency of differentiation was determined by FACS analysis of CSF1R and F4/80 surface antigen expression, using antibodies conjugated to phycoerythrin (PE-A) or allophycocyanin (APC-A). (B) BM-Mϕ were seeded in the upper compartment of a Boyden chamber, and the number of cells that migrated through the filter in response to CM from nonhypoxic (20% O2) or hypoxic (1% O2) MDA-MB-231 cells (BCCs), MSCs, or BCCs + MSCs in the lower compartment were counted under light microscopy after staining with crystal violet. (Scale bars: 0.2 mm.) Data were normalized to CM from BCCs at 20% O2. *P < 0.05 vs. 20% BCCs; **P < 0.01 vs. 20% BCCs; #P < 0.01 vs. 20% BCCs + MSCs. (C) BM-Mϕ migration in response to CM isolated from BCCs or BCCs + MSCs (supplemented with IgG or CCL5 NAb) was determined and normalized to CM from BCCs at 20% O2. (Scale bars: 0.2 mm.) *P < 0.05 vs. 20% BCCs; **P < 0.01 vs. 20% BCCs; #P < 0.05 vs. 20% IgG; ##P < 0.001 vs. 1% IgG. (D) BM-Mϕ migration in response to CM isolated from NTC or shCSF1-1 BCCs (alone or cocultured with MSCs) was determined and normalized to CM from NTC cells at 20% O2. (Scale bars: 0.2 mm.) *P < 0.05 vs. 20% NTC − MSCs; **P < 0.01 vs. 20% NTC − MSCs; #P < 0.05 vs. 1% NTC - MSCs; ##P < 0.001 vs. 1% NTC + MSCs.