Journal: Breast Cancer Research : BCR

Article Title: Dietary fat increases solid tumor growth and metastasis of 4T1 murine mammary carcinoma cells and mortality in obesity-resistant BALB/c mice

doi: 10.1186/bcr2927

Figure Lengend Snippet: Effect of various cytokines on proliferation (a), adhesion (b), and migration (c) of 4T1 cells in vitro . (a) 4T1 cells were plated in 24-well plates at 5 × 10 4 cells/well in DMEM supplemented with 100 ml/L FBS. One day later, the monolayers were serum deprived with DMEM supplemented with 10 ml/L charcoal-stripped FBS (serum-deprivation medium) for 24 h. After serum deprivation, the cells were incubated in serum-deprivation medium in the absence or presence of 29 ng/ml complement fragment 5a (C5a), 652 ng/ml soluble intercellular adhesion molecule (sICAM)-1, 190 ng/ml interleukin (IL)-16, 1,150 pg/ml macrophage colony-stimulating factor (M-CSF), 6.2 ng/ml tissue inhibitor of matrix metalloproteinase (TIMP)-1, and 970 pg/ml triggering receptor expressed on myeloid cells (TREM)-1 for 24 hours. Viable cell numbers were estimated. Each bar represents the mean ± SEM ( n = 3). Means without a common letter differ, P < 0.05. (b) Cells were plated in human collagen type I-coated CytoMatrix Cell Adhesion Strips and incubated with or without various cytokines (the identical concentrations of cytokines used in Figure 6a) in DMEM containing 10 ml/L charcoal-stripped FBS at 37°C for 45 min. The cells were stained with crystal violet, and the cell-bound stains were quantified by determining the absorbance at 570 nm. Each bar represents the mean ± SEM ( n = 7). Means without a common letter differ, P < 0.05. (c) Cells were serum deprived in DMEM supplemented with 10 ml/L charcoal-stripped FBS for 24 hours. Serum-deprived cells were plated onto the filter in 6.5-mm transwell inserts in 24-well plates at 5 × 10 4 cells/filter. Before the plating of the cells, the lower side of the transwell filter was precoated with type IV collagen. The lower chamber of the well was filled with DMEM containing 100 ml/L gelatinase-free charcoal-stripped FBS with or without various cytokines (the identical concentrations of cytokines used in Figure 6a). The cells were incubated for 16 hours. Migrated cells were stained with hematoxylin and eosin. Each bar represents the mean ± SEM ( n = 3). Means without a common letter differ, P < 0.05.

Article Snippet: Reagents were purchased from the following suppliers: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), Bouin solution, and anti-β-actin antibody from Sigma (St. Louis, MO, USA); antibodies against CD45, matrix metalloproteinase (MMP)-9, tissue inhibitor of matrix metalloproteinase (TIMP)-2, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1, and enzyme-linked immunosorbent assay (ELISA) kits for soluble ICAM-1, macrophage colony stimulating factor (M-CSF), TIMP-1, leptin, and triggering receptor expressed on myeloid cells (TREM)-1 from R and D Systems (Minneapolis, MN, USA); ELISA kits for complement fragment 5a (C5a) and interleukin (IL)-16 from USCN Life Science and Technology Company (Missouri City, TX, USA); antibodies against urokinase-type plasminogen activator (uPA) from Calbiochem (La Jolla, CA, USA); antibodies against E2F1, proliferating cell nuclear antigen (PCNA), p27, cyclin-dependent kinase (CDK)2, CDK4, cyclin A, cyclin D1, CD31, and vascular endothelial growth factor (VEGF), plasminogen activator inhibitor (PAI)-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Ki67 and F4/80 from Abcam (Cambridge, MA, USA); horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG from Amersham Biosciences (Arlington Heights, IL, USA); an antibody against CD68 from ABBIOTEC (San Diego, CA, USA); and Immobilon Western Chemiluminescent HRP Substrate and adhesion assay kit from Millipore Corporation (Billerica, MA, USA).

Techniques: Migration, In Vitro, Incubation, Staining