Fig. 5

TAM derived IL-6 augments migration and angiogenesis in breast cancer. A 4T1 cells were treated with CM of activated RAW264.7 cells or CM neutralized with IL-6 antibody (20 µg/ml). In separate experiments, 4T1 cells were pre-treated with Stattic (5 µM) followed by treatment with CM of activated RAW264.7. Cells were seeded in the upper chamber. In all groups, fetal bovine serum (10%) containing media was used in the lower chamber as chemoattractant. After 18 h, migrated cells were stained and quantified statistically using NIH-ImageJ software and displayed in the form of bar graph. The error bar represents mean ± SEM, *denotes p < 0.05, n = 3 independent experiments. B RAW264.7 cells were either treated with CM of 4T1 cells or pre-treated with SB203580 (10 µM) followed by treatment with CM of 4T1 cells and CM were collected after 12 h. In separate experiments, IL-6 was neutralized in CM of activated RAW264.7 cells by using IL-6 neutralizing antibody (20 µg/ml) and used for tube formation assay using HUVECs. In another setting, HUVECs were treated with recombinant IL-6 and tube formation assay was performed. After treatment, tubular structure was photographed and analysed. Number of junctions were measured by using AngioTool64 0.6a software and described as fold change compared to control in the form of bar graph. Error bars represent mean ± SEM, *denotes p < 0.05, **denotes p < 0.01 and ***denotes p < 0.001. C, D 4T1 cells were treated with either CM of activated RAW264.7 cells or CM neutralized with IL-6 antibody (20 µg/ml). In separate experiments, 4T1 cells were pre-treated with Stattic (5 µM) followed by treatment with CM of activated RAW264.7 cells. The media were replaced with fresh basal DMEM for 12 h and CM of 4T1 cells were collected and used for in vitro tube formation assay using HUVECs. After treatment, tubular structure was photographed and analysed. Number of junctions were measured using AngioTool64 0.6a software and expressed as fold change compared to control in the form of bar graph. Error bars represent mean ± SEM, *denotes p < 0.05, **denotes p < 0.01