Characterization of PKD Q4004X growth and selection.

<p>A: Telomerase activity assay and immune blot analysis of transduced and untransduced cells. Left panel: Telomerase activity detected as labeled telomerase product telomeric DNA ladders. DNA ladders are only observed in cells transduced with hTERT. Untransduced control cells or cells transduced with empty vector (pLXSN) have no detectable telomerase activity. Right panel: Exogenous telomerase expression confirmed by immune blot analysis. A 120 kDa band is observed in lysates made from PKD cells transfected with hTERT. <b>B: </b><b>Mutation detection in the PKD cell line.</b> A C to T mutation resulting in a premature truncation at amino acid 4004 is shown. <b>C:</b> Phase contrast image of untransduced cells (upper image) and hTERT transfected PKD cells (lower image) maintained in selection media. <b>D: </b><b>Fluorescence activated cell sorting of LTL and DBA labeled HK2 cells.</b> Fluorescence intensity at 575 nM/unit cell area versus fluorescence intensity at 530 nM/unit cell area are plotted on the Y and X axis respectively. Since DBA was tagged with rhodamine, cells labeled with the DBA marker are expected to register in the region demarcated by the circle. <b>E: </b><b>Fluorescence activated cell sorting of LTL and DBA labeled MDCK-II cells.</b> Fluorescence intensity at 575 nM/unit cell area versus fluorescence intensity at 530 nM/unit cell area are plotted on the Y and X axis respectively. Since DBA was tagged with rhodamine, cells labeled with the DBA marker are expected to register in the region demarcated by the circle and shown in green. MDCK-II cells are a mixed population basted on this assay (compare red and green populations). <b>F: </b><b>Fluorescence activated cell sorting of LTL and DBA labeled PKD Q4004X cells.</b> Fluorescence intensity at 575 nM/unit cell area versus fluorescence intensity at 530 nM/unit cell area are plotted on the Y and X axis respectively. Since DBA was tagged with rhodamine, cells labeled with the DBA marker are expected to register in the region demarcated by the circle. Few cells have DBA labeling with greater than 99% of the cells showing LTL positive labeling.