Soluble factor(s) from MDA-MB-231 stimulate MCF-10A to increase production of IL-6. A. MCF-10A cells were treated with MDA-MB-231 conditioned media for 24, 48, or 96 hours. After this treatment time, MCF-10A cells were washed and allowed to produce their own cytokines for 24 hours, after which ELISA analysis was performed to assay for IL-6 secretion. For the 0 hour treatment, conditioned media was added, but was washed away immediately and collected for analysis. This was done to show that any soluble factors from the MDA-MB-231 conditioned media were washed away and were not contaminating our results. For untreated MCF-10A cells, the cells were mock-treated with fresh 2% FBS, 1× DMEM instead. B. To confirm that soluble factors from the MDA-MB-231 conditioned media were inducing MCF-10A to produce IL-6, RNA collected from these cells and used to perform non-quantitative RT-PCR. RNA from MDA-MB-231 was used as a positive control. 231: MDA-MB-231; No Tx: no treatment; CM: conditioned media.