Figure 7. Effect of PTK7 RNAi knockdown on ATRT tumor cells in culture.

A. Real time PCR using primers to PTK7 and GAPDH indicate knockdown of PTK7 in ATRT cell lines ATRT-05 and ATRT-06. Relative gene expression of PTK7/fold change expression levels was calculated using the ΔΔCq method, using Ct values obtained with the PTK7 primers and the housekeeping gene GAPDH and plotted in the different siRNA treatments (B). Multiple cell viability assays (n=3) indicate that transfection of ATRT-05 and ATRT-06 with PTK7 RNAi significantly reduces the growth of ATRT cell lines as compared to untransfected controls (abbreviated Con. untreated), according to a two-tailed t test p<0.01 (**), while transfection of PTK7 RNAi (abbreviated PTK7siRNA) does not affect the viability of control HEK-293 cells. There is no significant difference between viability of nontransfected controls and the scrambled negative nonspecific control (abbreviated scrambled). C+D. Images of the viability of ATRT cell line ATRT-05 48 hours following RNAi transfection with PTK7 RNAi. (D) indicates the presence of very few cells following PTK7 siRNA as compared to mock transfected cells (C). Cells were stained with DAPI and visualized with DIC (10x). Scale bar= 100 microns. Transfection with PTK7 siRNA in ATRT cell lines resulted in a loss of PTK7 immunoreactivity (F) as compared to controls (E). Hoechst staining was performed to visualize nuclei in ATRT cell lines following knockdown of PTK7 (H) as compared to controls (G). Knockdown of PTK7 induced caspase-3 immunoreactivity (J) as compared to controls ().