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Fig. 4. Onco toxicity and translocation are enhanced by endosome neutralization. (A) Cytotoxicity to HeLa and A431 cells. Cells were pre-incubated for 30 minutes with 0.25 µM monensin, 20 mM NH₄Cl (Amm Cl) or 100 nM bafilomycin (Baf), as indicated, before adding various concentrations of Onco. Cell viability was measured after 24 hours (HeLa) or 36 hours (A431). The results are expressed as the Onco concentration generating a growth inhibition of 50% (IC₅₀). Mean ± s.e.m. of at least three separate experiments. (B) Endosomal pH measurements. A431 cells were labeled for 40 minutes with Fl-Tf-Rh before acquiring Fl and Rh fluorescence images using a confocal microscope. The Fl/Rh intracellular intensity ratio was then used together with a calibration curve to determine the endosomal pH (Dunn et al., 1994; Presley et al., 1997) in living cells treated with the indicated drug. (C) Endosome neutralization enhances Onco translocation. ¹²⁵I-Onco- or ¹²⁵I-Tf-loaded lymphocyte endosomes were purified and resuspended in translocation buffer supplemented with 10 mM ATP. Translocation assays (Morlon-Guyot et al., 2003; Vendeville et al., 2004) were performed for 1 hour in the presence or absence of 400 nM Baf or 20 µM monensin, as indicated. Translocation activity corresponds to the time course increase in the supernatant/(endosome plus supernatant) radioactivity ratio. (D) Onco translocation requires cytosolic ATP hydrolysis. Translocation was assayed in the presence of Baf and 10 mM ATP or ATPS, as indicated.

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