Figure 1.

MDA-MB-231 tumour cells influence the regulation of MMP1, type I collagen and Smad7 gene expression in CCD-1068SK fibroblasts during direct co-culture. For direct co-culture experiments, CCD-1068SK fibroblasts were labelled with green fluorescent dye, PKH67, mixed with an equal number of MDA-MB-231 breast tumour cells and co-cultured for 48 hours in serum-free medium. Fibroblasts were separated from tumour cells by FACS and used for further analysis. CCD-1068SK monocultures were used as controls. (A) Quantitative real-time RT-PCR analysis of MMP1, COL1A1, COL1A2, CCN2 and Smad7 mRNA levels in co-cultured CCD-1068SK fibroblasts. The graph shows the mean ± SD from a representative experiment, relative to GAPDH (*p ≤ 0.05, n = 3). (B) Western blotting results show endogenous α1(I) and α2(I) procollagen, CCN2 and Smad7 protein levels in CCD-1068SK fibroblasts after direct co-cultures. β-tubulin was used as a loading control. (C) De novo synthesis of type I collagen measured by [3H]-proline incorporation into CCD-1068SK/MDA-MB-231 direct co-culture medium. CCD-1068SK fibroblasts monocultures and co-cultures with non-tumourigenic MCF12A cells were used as controls. The medium was run on an 8% SDS-PAGE gel and exposed to film for 7 days at -80°C, before being developed. (D) Quantitative real-time RT-PCR of indirectly co-cultured cells as described show relative COL1A1, COL1A2, CCN2 and Smad7 mRNA levels in CCD-1068SK fibroblasts. The graph shows the mean ± SD from a representative experiment (n = 3). (E) Western blotting results show endogenous α1(I) and α2(I) procollagen levels, CCN2 and Smad7 in CCD-1068SK fibroblasts after indirect co-culture with tumour cells. β-tubulin was used as a loading control. Abbreviations: CCD, CCD-1068SK; MDA, MDA-MB-231. Brackets enclose the cell line with which CCD-1068SK fibroblasts were co-cultured.