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sc-12-0619_sm_SupplFigure1.TIF99KSupplemental Figure 1. RAW macrophages possess an M2 phenotype. RAW macrophages were cultured either alone, co-cultured with 4T1 breast cancer cells or stimulated with INF- or IL-13 for 24 h, and then analyzed by flow cytometry. Macrophages were identified by CD45+/F4/80+. RAW macrophages are CD206high, IL-10high, CD86low, MHC Class II low, characteristic of M2 macrophages and TAMs.
sc-12-0619_sm_SupplFigure2.TIF151KSupplemental Figure 2. The pattern of cytokines and growth factors released in TAM (M2-macrophages). (A) The release of Th2 cytokines (IL-4, IL-10 IL-6 and TNF-α) and growth factors (EGF, VEGF and TGF-β1) were increased in TAMs derived from 4T1 tumor tissue, reversely, Th1 cytokines (IL-2, IL-7, IL12a, IL-12b and INF-r) were released largly in MI macrophages derived from spleen of normal mice. (B-C) The expression of cytokines (IL-4, IL-10 IL-6 and TNF-α) and growth factors (EGF, VEGF and TGF-β1) were up-regulated in RAW cells after co-cultured with 4T1 tumor cells or 4T1 tumor conditional medium. (D) The expression of growth factors (TNF-α, EGF, VEGF and TGF-β1) were up-regulated in 4T1 cells after co-cultured with RAW cells.
sc-12-0619_sm_SupplFigure3.TIF113KSupplemental Figure 3. 4T1 and 4T07 breast cancer cells contain a side population (SP) with cancer stem cell-like properties. (A) 4T07 or 4T1 breast cancer cells were first stained with Hoechst 3334 dye, and then reacted with either Sca-1 or ABCG2 antibodies labeled with PE to determine the SP and its expression of Sca-1 and ABCG2. (B) SP of 4T07 cells was sorted by Flow Cytometry after being stained with Hoechst 3334 dye, and a group of Balb/c mice were implanted in the cleared fat pad with either the SP or Non-SP fraction of 4T07 breast cancer cells at 7×103 per mouse. (C) Tumor volume was measured every 3-5 days. (D) All mice were sacrificed on day 25 after tumor cell implantation and analyzed for the metastasis score on lungs

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