Figure 1.

Induction of NF-κB in fibroblasts by hormone-independent breast cancer cell-derived CM. (A) NF-κB activation in fibroblasts. Six micrograms of whole-cell extract from HLF-1 treated with CM from indicated cell lines for 3 hr (Upper) or 24 hr (Lower) was subjected to EMSA by using NF-κB probe. NF-κB activation by TNFα (10 ng/ml) is also shown as a control. Presence of the NF-κB subunit RelA in DNA–protein complex was verified by antibody supershift assay. In this assay, whole-cell extracts from HLF-1 treated with MDA-MB-231 CM was incubated with either NF-κB probe alone (lane 9) or probe with an antibody against RelA (lane 10). The DNA–protein complex supershifted by the antibody is indicated (ss). (B) CM from breast cancer cells do not effect DNA binding of transcription factors AP-1 and CTF/NF-1. HLF-1 was treated with CM from indicated cell lines for 3 hr, and 6 μg of whole-cell extract was subjected to EMSA by using NF-κB probe (Top), AP-1 probe (Middle), and CTF/NF-1 probe (Bottom). (C) Induction of NF-κB in normal fibroblasts. Fibroblasts isolated from a skin biopsy were treated with CM from indicated cell lines for 3 hr and analyzed for NF-κB DNA-binding activity. (D) Protease/proteosome inhibitors prevent NF-κB activation by MDA-MB-231 CM. HLF-1 was incubated with indicated concentrations of TPCK or MG132 for 1 hr before the addition of MDA-MB-231 CM. Whole-cell extract was prepared 3 hr after the addition of CM and subjected to EMSA. (E) Induction of NF-κB in macrophages and endothelial cells by breast cancer cell CM. THP-1 cells (macrophages, lanes 1–6) and bovine heart endothelial cells (lanes 7–10) were incubated with CM from indicated cell lines for 24 hr. Whole-cell extracts were analyzed for NF-κB as above.