Figure 2.

HRP as a reporter for endo/lysosomal escape in isolated organelles. Lysosomes were isolated by cell fractionation form ECV-304 cells (3 × 10⁷ cells). (A) First, the permeabilizing effects of SA1641 on the lysosomal membranes were evaluated by the β-N-acetylglucosaminidase (NAG) release assay.The permeabilizing effects of digitonin (highly lytic saponin) and α-hederin (non-lytic saponin) were simultaneously determined as controls; (B) Thereafter, the endo/lysosomal escape enhancement of saporin-HRP observed in the cytotoxicity assay in Figure 1F was tried, to monitor in isolated lysosomes loaded with saporin-HRP (from cells previously treated with the conjugate at 100 nM and 37 °C for 6 h) by determination of the peroxidase activity. Saponin concentrations are shown in µM to allow a better comparison. Each data point represents the mean ± SD, n = 3.