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. 2014 May 22;6(5):1644–1666. doi: 10.3390/toxins6051644

Figure 5.

Figure 5

Ricin A-chain (RTA) as a reporter for endo/lysosomal escape. The report of the endo/lysosomal escape enhancement of saporin-KQ (an enzymatically inactive saporin in order to avoid interferences in the readout of the experiment) was attempted by measuring the cytotoxic activity of RTA. (A) Saporin-KQ and RTA were chemically cross-linked. The reaction mixture was analysed by SDS-PAGE, and both unconjugated saporin-KQ and RTA were included as controls; (B) Saporin-KQ-RTA was purified by Ni-NTA chromatography. Proteins were eluted with increasing concentrations of imidazole (31, 62, 125 and 250 mM), and an SDS-PAGE analysis was effectuated for the fractions.Saporin-KQ-RTA was eluted in Fractions 5 at 125 mM and 1–5 at 250 mM imidazole (see arrows); Cytotoxicity of (C) saporin; (D) RTA; (E) saporin-KQ; and (F) saporin-KQ-RTA in combination with SO1861. ECV-304 (4000 cells/well) were treated with saporin, RTA, saporin-KQ or saporin-KQ-RTA in a concentration range from 0.01 to 1000 nM in the presence or absence of SO1861 (final concentration of 1–2 µg/mL) for 48 h. The viability of cells was evaluated by the MTT assay. Data is the mean ± SD, n = 4.