Recombinant il 34

MCF7, SK-BR-3 and MDA-MB-231 breast cancer cell lines were starved over night in serum-free DMEM medium. Breast cancer cells (1×10⁵) in DMEM with 2% FBS were added to the top of each Boyden migration chamber (8 μm, 12-well plate format; BD Biosciences, Palo Alto, CA) with or without CSF-1R-blocking antibody (10μg/ml) followed by treatment with 200 ng/ml recombinant IL-34 or CSF-1 protein (R&D Systems, McKinley Place, MN, USA). After 48 h, medium was removed and membranes were processed as described [59 (link)].

Recombinant IL-34 was purchased from R&D Systems (Minneapolis, USA). The IL-17 inhibitor plumbagin (PB) was purchased from Novus (Centennial, USA). PB (10 mg)was slowly dissolved in 530 µl of anhydrousdimethyl sulfoxide (DMSO) by gentle vortexing and administered to cells in media containing the solvent at a final concentration of 4 µM. A cell proliferation and cytotoxicity assaykit was purchased from Biyuntian Reagent Company (Nantong, China). A FITC Annexin V-FITC/PI apoptosis detection kit was purchased from BD Biosciences (New York, USA). Dulbecco’s modified Eagle’s medium (DMEM) and 10% fetal bovine serum (FBS) were purchased from HyClone (Utah, USA).