Journal: Journal of Cellular and Molecular Medicine

Article Title: The Overexpression of Collagen Receptor DDR1 is Associated With Chromosome Instability and Aneuploidy in Diffuse Large B‐Cell Lymphoma

doi: 10.1111/jcmm.70318

Figure Lengend Snippet: Activated DDR1 downregulates expression of the mitotic kinesin, CENPE . (A) Upper panels show the significant overlap between genes upregulated by DDR1 in normal GC B cells and those upregulated in either GCB‐ or ABC‐DLBCL compared with normal GC B cells (total genes on both platforms = 26,438). The lower panel shows GO analysis of genes upregulated by DDR1 in normal GC B cells and also upregulated in both GCB and ABC‐DLBCL. Open bars indicate the percentage of genes present in the GO term that would be expected to be observed in the regulated gene list by chance. Shaded bars indicate the percentage of genes in the regulated list with a given GO term that were actually observed. (B) RT‐qPCR confirming that collagen treatment of DDR1‐expressing GC B cells significantly reduced the mRNA levels of CENPE . Shown are triplicate data on tonsillar GC B cells isolated from three different donors (T20, T21 and T42). (C) Compared to empty vector (EV) control, the addition of collagen significantly reduced CENPE mRNA expression in DDR1‐expressing B‐cell lines, BJAB (maximally after 6 h of stimulation) and DG75 (maximally after 2 h of stimulation). Data are representative of three separate biological replicates. (D) Activation of an ectopically expressed DDR1 reduced CENPE protein expression in both BJAB and DG75 cells. An antibody specific for DDR1 phosphorylated on Tyr792 was used to confirm activation of DDR1. As expected, the activation of DDR1 also reduced total DDR1 levels. β‐Actin was used as a loading control. Data shown are representative of three separate biological replicates. (E) A constitutively activated DDR1 construct also downregulated CENPE protein expression. Shown here are the mean fluorescence intensities for DDR1 and CENPE in DG75 cells transfected with the constitutively active DDR1 gene. Cells expressing DDR1 were significantly more likely to lack CENPE protein expression than were untransfected cells in the same population ( p < 0.0001). (F) Immunohistochemistry showing the downregulation of CENPE protein in DLBCL tumour cells. Top left panel shows strong expression of CENPE protein in a normal germinal centre (GC) of tonsil. Original magnification ×200. Remaining panels show examples of staining in a case in which CENPE was not detected in tumour cells (top right), a case with weak expression in tumour cells (bottom left) and a case showing strong staining (bottom right). Original magnification ×400. Black arrows show tumour cells. Red arrows indicate nonmalignant cells that are positive for CENPE.

Article Snippet: DG75 and BJAB B‐cell lymphoma lines (DSMZ, Braunschweig, Germany) were cultivated in RPMI 1640 (Gibco; Life Technologies Ltd., Paisley, UK) culture media, with 10% FCS and 1% penicillin–streptomycin (Gibco).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Plasmid Preparation, Control, Activation Assay, Construct, Fluorescence, Transfection, Immunohistochemistry, Staining