Figure 7.

ZNF nuclease-mediated targeting of SPHK1 in MDA-MB-231 and cytotoxic sensitivity to SphK inhibitors. MDA-MB-231 cells were transiently-transfected with a custom SphK1 ZNF nuclease genomic targeting cassette along with CMV–eGFP expressing plasmid followed by FACS-mediated cell sorting of cell pools of varying fluorescence intensity (P1, P2 and P3) and subsequent culture-expanded pools (P2.e and P3.e). Genomic PCR was performed to assess genomic modification of the SPHK1 locus in various transfected and expanded MDA-MB-231 cell pools. Positive genomic alteration is noted by a black arrow corresponding to a PCR products band at ∼150–160 nt by gel electrophoresis (a). To assess MDA-MB-231 cell viability, cells were seeded and treated with varying concentrations of DMS, SKI II, FTY720 and SEW2781 for 7 days followed by cell viability determination by Cell Titer Glo analysis. Table IC50 calculations were based on the concentration response values analyzed by four-parameter non-linear regression curve fitting using GraphPad Prism (ver6.0) from two independent studies (b).