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Advancements in the Development of HIF-1α-Activated Protein Switches for Use in Enzyme Prodrug Therapy

Figure 5

Characterization of the effects of Haps59 expression in Flp-In 293 cells and E. coli.

(A) Western blot with anti-HIF-1α antibodies showing that the addition of CoCl2 causes the accumulation of HIF-1α in Flp-In 293 cells. See Figure S5 for full blot. (B) Toxicity of 5FC to stable isogenic pools of Flp-In 293 cells containing EV, yCD, and Haps59. Cells were grown in the absence (i.e. −HIF-1α) or presence (i.e. + HIF-1α) of 50 µM CoCl2, and increasing concentrations of 5FC. Percent survival was calculated by measuring the total DNA of surviving cells in each well and normalizing between wells containing no 5FC (i.e. 100% survival) and wells containing no cells (i.e. 0% survival). Error bars represent the standard deviation across three replicates. (C) Dot toxicity assay indicates that CITED2 but not E1A activates Hap59 in E. coli. Equal density dilutions of log phase cultures of strains expressing Haps59 and either the p300 binding fragments of HIF-1α, CITED2, or E1A were plated on increasing concentrations of 5FC.

Figure 5