Exogenous IL-6 enhances pStat3 levels, binding activity and cell migration in MCF10A-Ras cells. (a) Extracts from MCF10A-Ras cells (C) and MCF10A-Ras treated with IL-6 (10ng/ml) for one hour (IL-6) were analyzed by Western blot analysis for pStat3, Stat3 and Tubulin (b) Human IL-6 mRNA levels from MCF10A-Ras cells (C) and MCF10A-Ras cells treated with IL-6 for four hours (IL-6) were determined by RT-PCR and normalized to β-Actin. (c) EMSA was performed with nuclear extracts from the cell lines described in a. Stat3 DNA binding was supershifted with anti-Stat3 antibody (IL6+Ab). (d) MCF10A-Ras cells (C) or Stat3 shRNA (S3sh) were plated into Boyden chambers and cell migration was determined in the absence or presence of IL-6 (C+IL6, S3Sh+IL-6) with crystal violet staining after 24 hrs. Results are expressed as cells/field (mean ± SD of triplicates from three independent experiments).