We then examined the phenotype of the selected transfected cells after cloning by limit dilution. Our results,indicating that NRP 152 and BPH 1 cells underwent changes in Rapamycin order phenotype consistent with that of malignant cells,are selleck presented here. Results Selection of Transfected NRP 152 and BPH 1 Cells Two weeks after transfection selleck chem inhibitor with either pIRES or pIRES S3c and selection with G418,no surviving Inhibitors,Modulators,Libraries cells were observed in the wells that received Clonfectin only. Growth of cells was observed in Inhibitors,Modulators,Libraries all wells that received either of the Inhibitors,Modulators,Libraries plasmids plus Clonfectin. Transfected cells were expanded for further analysis in complete medium. A summary of cells and clones and what their phenotypes were is given in Table 1.
To summarize briefly,since the full results will be discussed in this section,we observed the following changes.
NRP Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries 152 cells require a variety of growth factors and addi tives in their medium,152 pIRES cells required the same medium Inhibitors,Modulators,Libraries as NRP 152 cells. But 152 S3c cells grew in DMEM Hams F12 supple mented only with 10% newborn calf serum. Moreover,152 S3c cells expressed EGFP and the FLAG epitope,which is part of the S3c gene. Both 152 pIRES and 152 S3c cells grew in the pres ence of G418. BPH 1 cells grow in RPMI 1640 Inhibitors,Modulators,Libraries supplemented with bovine serum,therefore this line does not have growth factor dependence to begin with. BPH pIRES and BPH S3c cells,aside from exhibiting G418 resistance,expressed EGFP,but only BPH S3c expressed the FLAG epitope of the S3c gene.
The evidence for these observations given Inhibitors,Modulators,Libraries in Table 1 is presented in the rest of this Inhibitors,Modulators,Libraries section.
Expression of FLAG and EGFP in 152 S3c and Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries BPH Inhibitors,Modulators,Libraries S3c Cells Was Observed After Transfection and Selection with Antibiotics After no viable cells were observed following antibiotic Inhibitors,Modulators,Libraries treatment,we analyzed transfected cells Inhibitors,Modulators,Libraries for the presence of the markers flanking the S3c gene on the plasmids used,FLAG and EGFP. The analyses were done by flow cytometry on a FACScan,also by Western blot using spe cific Abs,and the results are presented in Figure 2.
In Pan els A through D,the mean fluorescence intensities of representative clones of 152 S3c and BPH S3c cells stained with monoclonal Ab to FLAG plus fluorescent goat anti mouse F,as well as the enhanced green flu orescent protein fluorescence intensities of transfected cells,are shown.
Panel A displays the anti FLAG fluores cence intensity of 1 representative Inhibitors,Modulators,Libraries clone of 152 S3c compared to untransfected selleck inhibitor NRP 152 cells,approximately this explanation 95% of enzyme inhibitor the 152 S3c cells stained with the anti FLAG antibody. Similary,Panel B shows the fluorescence intensity of anti FLAG stained BPH 1 cells compared to anti FLAG stained BPH S3c clone,where approximately 76% of the BPH S3c cells stained with the anti FLAG antibody. Panels C and D display the EGFP fluorescence for clones of 152 S3c and BPH S3c cells,compared with untransfected cells,respec tively.