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. Author manuscript; available in PMC: 2011 Feb 1.

Published in final edited form as: Nat Med. 2010 Jul 18;16(8):921–926. doi: 10.1038/nm.2185

Testing the TK PCA in separate cell lines and PPI systems. (a) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin) in different cell lines, as measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, SKBr3 cells, and SKOV3 cells. The error bar is the standard error of the mean for three samples. There was a statistically significant increase in measured TK activity upon addition of rapamycin to all 3 cell lines. Despite lack of normalization of transfection efficiency, there was a similar approximate 3-fold rise in split-TK complementation upon PPI regardless of which cell line was used. Western blot analysis of all 3 cells using antibody to TK before and after addition of rapamycin shows adequate expression levels. (b) A vector was constructed that transiently expresses a fusion protein with split TK fragments (containing a V119C point mutation) and substituting HIF1α for FRB, and VHL for FKBP12. Each construct was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently co-transfected (200 ng DNA each vector per well) to express nTK(V119C)-HIF1α and VHL-cTK, an empty vector, and full length HSV1-sr39TK as a positive control for 48 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with increasing doses of desferrioxamine (DFO) resulted in a proportional reduction in interaction of HIF1α and VHL. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to TK after treatment with the different doses of DFO shows adequate expression levels in 293T cells. (c) Schematic diagram of the intramolecular folding sensor construct with the split TK fragments on either side of the estrogen receptor ligand binding domain (ER–LBD), and each construct was made by cloning into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently transfected (500 ng DNA per well) to express the intramolecular folding sensor and treated with the indicated ER ligands or carrier control (dymethyl sulfoxide, DMSO) for 24 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with the ER ligands 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), raloxifene (RAL), methyl piperidinylethoxy pyrazole (MPP), genistein (GEN), diethylstilbestrol (DES), and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) led to levels of intramolecular-folding-assisted complementation that were significantly higher than that of carrier control-treated cells (P < .05) except for genistein. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to ERα after treatment with the different ligands shows adequate expression levels in 293T cells.

Testing the TK PCA in separate cell lines and PPI systems. (a) Graph to show comparison of coexpressed chimeras carrying nTK or cTK with FRB/FKBP12 (with and without rapamycin) in different cell lines, as measured by TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein) in transiently transfected 293T cells, SKBr3 cells, and SKOV3 cells. The error bar is the standard error of the mean for three samples. There was a statistically significant increase in measured TK activity upon addition of rapamycin to all 3 cell lines. Despite lack of normalization of transfection efficiency, there was a similar approximate 3-fold rise in split-TK complementation upon PPI regardless of which cell line was used. Western blot analysis of all 3 cells using antibody to TK before and after addition of rapamycin shows adequate expression levels. (b) A vector was constructed that transiently expresses a fusion protein with split TK fragments (containing a V119C point mutation) and substituting HIF1α for FRB, and VHL for FKBP12. Each construct was cloned into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently co-transfected (200 ng DNA each vector per well) to express nTK(V119C)-HIF1α and VHL-cTK, an empty vector, and full length HSV1-sr39TK as a positive control for 48 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with increasing doses of desferrioxamine (DFO) resulted in a proportional reduction in interaction of HIF1α and VHL. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to TK after treatment with the different doses of DFO shows adequate expression levels in 293T cells. (c) Schematic diagram of the intramolecular folding sensor construct with the split TK fragments on either side of the estrogen receptor ligand binding domain (ER–LBD), and each construct was made by cloning into a pcDNA3.1 (+) plasmid backbone, under control of a CMV promoter. 293T cells were transiently transfected (500 ng DNA per well) to express the intramolecular folding sensor and treated with the indicated ER ligands or carrier control (dymethyl sulfoxide, DMSO) for 24 h, followed by measurement of TK enzyme uptake (expressed as normalized dpm of cells/dpm in medium/microgram of protein). Treatment with the ER ligands 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), raloxifene (RAL), methyl piperidinylethoxy pyrazole (MPP), genistein (GEN), diethylstilbestrol (DES), and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) led to levels of intramolecular-folding-assisted complementation that were significantly higher than that of carrier control-treated cells (P < .05) except for genistein. The error bar is the standard error of the mean for three samples. Western blot analysis using antibody to ERα after treatment with the different ligands shows adequate expression levels in 293T cells.