Figure 7

NF-κB is essential in mediating TNF-α-induced release of chemokines by MSCs. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 15 minutes. The levels of IκBα (the negative regulator of the NF-κB pathway) were determined by WB analyses. GAPDH was used as a loading control throughout. (B) CAFs were generated by culturing MSCs with Tumor CM from MDA-MB-231 (MDA) or MCF-7 breast tumor cells over a prolonged period of time (~30 days). TNF-α (50 ng/ml) was added for the last 24 hours to MSCs + Tumor CM cells and IκBα levels were determined by WB analyses. (C) CAF #1 cells were stimulated for 48 hours by TNF-α (50 ng/ml). IκBα levels were determined by WB analyses. (D) Human BM-derived MSCs were stimulated with TNF-α (50 ng/ml) for 10 minutes. p65 phosphorylation was determined by WB analyses. (E) Human BM-derived MSCs were transiently transfected by siRNA to p65 or by control siRNA. (E1) p65 expression was determined by WB analyses. (E2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 48 hours. Expression of CCL2 and CXCL8 in the supernatants of the cells was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.