Restoration of ROR2 expression impairs tumour growth in vitro and in vivo. The effect of ROR2 restoration was studied in the DLD1 colon cancer cell line. (A) WB showing restoration of ROR2 after transfection with the pcDNA3.1-ROR2-Flag construct; anti-Flag antibody was used. (B) MTT analysis of cell viability in DLD1 mock-(squares) and ROR2-Flag-transfected (circles) cells. (C) Colony formation assay of DLD1-transfected cell line. DLD1 mock-and ROR2-Flag-transfected cells were seeded in six-well plates and cultured for two weeks, when colonies were visible. Colony number is expressed relative to number of cells seeded. (D) Quantitative RT-PCR analysis of ROR2 mRNA relative to GAPDH in the SW480 cell line transfected with a control siRNA (control iRNA) or a ROR2-specific siRNA (ROR2 iRNA). (E) MTT analysis of viability in SW480 cells expressing ROR2 (control iRNA, squares) or ROR2 knocked-down (ROR2 iRNA, circles). (F) Effect of restoration of ROR2 expression on in vivo tumour growth. Mouse xenograft model, in which DLD1 mock-transfected cells were injected subcutaneously into the left flank and ROR2-transfected cells into the right flank of athymic nude-Foxn1nu/un mice; representative images are shown (left). The right panels shows quantification of tumour size (cm3) in the mouse xenograft model; DLD1 mock-(circles) and ROR2-transfected cells (squares). (** p = < 0.01).