IL-6 is required for tumorigenesis of Ras transformed MCF10A cells. (a) Ras transformed MCF10A cells expressing control (C) or IL-6 shRNA (IL-6sh) were stimulated with TNF-α and analyzed for levels of IL-6 and normalized to actin by RT-PCR. (b) Extracts from MCF10A-Ras (RasC) and MCF10AIL6Sh (RasIL6sh) were analyzed for Stat3, pStat3, Ras and Tubulin levels. (c) MCF10A-Ras (RasC) and MCF10AIL6Sh (RasIL6Sh) cells were plated in six-well dishes and cell numbers were determined daily for seven days. Additionally, RasC cells were treated with IL-6 (10 ng/ml) (Ras+IL6) daily and cell numbers were determined. Each data point represents the mean value from triplicate wells. (d) MCF10A-Ras (C) and MCF10AIL6Sh (IL6Sh) cells 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). (e) Tumor growth in nude mice using MCF10A-Ras cells expressing control (C) or IL-6 shRNA (IL-6sh) was determined (mean ± SD from five independent injections).