Fig. 4

TAM derived IL-6 enhance stemness in breast cancer through STAT- 3 pathway. A 4T1 cells were treated with either CM of activated RAW264.7 cells or CM of activated RAW264.7 cells neutralized with IL-6 antibody (20 µg/ml) and expression of p-STAT-3 was examined by western blotting. STAT-3 was used as loading control. B, C In similar conditions, expression of p-STAT-3 was examined by immunofluorescence in 4T1 cells. Bar graphs represent corrected total cell fluorescence (CTCF) of p-STAT-3 as quantified by ImageJ software, mean ± SEM, *denotes p < 0.05, n = 3 independent experiments. D, E 4T1 cells were either untreated or treated with Stattic (5 µM), a STAT-3 inhibitor, and then treated with CM of activated RAW264.7 and CSC phenotype was examined by analysing Sca-1 expression and ALDH1 activity using flow cytometry. F 4T1 cells were either untreated or treated with Stattic (5 µM), a STAT-3 inhibitor, and then treated with CM of activated RAW264.7 and expression of p-STAT-3, Sox-2, Oct-3/4 and Nanog were analysed by western blotting. STAT-3 and Actin were used as respective loading control. Densitometry analysis was performed using NIH-ImageJ software. Relative fold changes with respect to control are shown