Fig. 1

Breast Cancer Conditioned Media (CM) activate macrophages and enhances IL-6 expression. A Macrophages (RAW264.7 cells) were treated with CM of 4T1 cells for 24 h and expression of CD206 was examined by BD FACSCanto II analyser. B RAW264.7 cells were either cultured alone or treated with CM of 4T1 for 12 h and CM were collected and analysed for secretomics by Orbitrap Fusion mass spectrometer (ThermoScientific™, USA). Heatmap showing the differentially expressed secretory proteins in control and CM of activated macrophage C Bar graph showing fold increase in IL-6 expression in CM of activated macrophages compared to control. Error bars represent mean ± SEM, ***denotes p < 0.001, n = 3 independent experiments. D Bar graph represents the relative fold change in IL-6 expression in activated RAW264.7 cells compared to control by qRT-PCR analysis and calculated using ΔΔCt method. β-actin served as endogenous control for normalization. Error bars represent mean ± SEM, *denotes p < 0.05, n = 4 independent experiments. E Bar graph represents IL-6 concentration in CM of activated RAW264.7 cells vs control as examined by ELISA. Error bars represent mean ± SEM, ***denotes p < 0.001, n = 4 independent experiments. F RAW264.7 cells were treated with CM of 4T1 for 24 h. Brefeldin A was added to inhibit the secretion of IL-6. After treatment, cells were processed and analysed for IL-6 expression using FACS Canto II analyser. G Bar graph represents the relative fold change in IL-6 expression by q-PCR analysis in TAMs isolated from 4T1 breast tumors using F4/80 marker by FACS sorting. Peritoneal macrophages were used as control. β-actin served as endogenous control