Fig. 2

Breast cancer cells induce p-38 mediated AP-1 dependent IL-6 expression in tumor activated macrophages. A RAW264.7 cells were treated with CM of 4T1 cells and expressions of p-p38, c-Fos and c-Jun were analysed by western blot. Actin was used as loading control. Densitometry analysis was performed using NIH-ImageJ software. Relative fold changes with respect to control are shown. B-D RAW264.7 cells were treated with CM of 4T1 cells and expressions of p-p38, c-Jun and c-Fos were analysed by immunofluorescence study. Bar graphs represent corrected total cell fluorescence (CTCF) of p-p38, c-Jun and c-Fos respectively as quantified by ImageJ software, mean ± SEM, *denoted p < 0.05, n = 3 independent experiments). E Binding of AP-1 to IL-6 promoter upon CM of 4T1 treatment. RAW264.7 cells were treated with CM derived from 4T1 cells and ChIP analysis was performed by immunoprecipitation with c-Jun antibody and PCR amplified using AP-1 specific primers. F Bar graph represents the relative fold change in IL-6 expression in RAW264.7 cells treated with 4T1 CM with or without pre-treatment with SB203580 compared to control by qRT-PCR analysis and calculated using ΔΔCt method. β-actin served as control for normalization, mean ± SEM, **denotes p < 0.01, n = 5 independent experiments. G Bar graph represents IL-6 concentration in CM of RAW264.7 cells treated with 4T1 CM with or without pre-treatment with SB203580 by ELISA, graph represents 1 of 3 technical repeats