Figure 6.

Induction of DCN expression in BGN transfectants. A. Determination of DCN expression in BGN^high HER-2/neu⁺ cells. Relative mRNA and protein expression of DCN were determined by qPCR and Western blot, respectively. DCN mRNA expression was analyzed in the NIH3T3 BGN^low/neg and BGN^high HER-2/neu⁺ cells and transcription was correlated to NIH3T3 cells (set 1). For protein expression, 50 µg protein/cell line was separated by 10% SDS-Page, transferred onto a nitrocellulose membrane, before immunostaining was performed with an anti-DCN-specific mAb as described in Materials and Methods. Staining of the Western blot with an anti-β-actin-specific mAb served as loading control. B. Downregulation of DCN expression by HER-2/neu overexpression in human tumor cells. The mRNA and protein expression of DCN was determined in human HER-2/neu model systems as described in Materials and Methods. C. Enhanced expression of DCN in BGN^high HER-2/neu⁺ cells in vivo. DCN mRNA and protein expression was determined in BGN^low and BGN^high HER-2/neu⁺ tumors as described in Materials and Methods. D. Increased MHC class I surface expression in the presence of DCN and/or BGN. Cells were treated with recombinant DCN (1.5 µg/mL) alone or in combination with recombinant (1 µg/mL) BGN for 23h, before MHC class I expression of untreated DCN and DCN/BGN-treated cells was determined by flow cytometry using an anti-H-2L^q mAb. MFI of untreated NIH3T3 cells and BGN^low HER-2/neu⁺ cells was set 100%.