Figure 4. SLO-induced endosomes mature into acidic compartments, where SLO is degraded by lysosomal enzymes.

A and B) Cells were incubated with GFP-SLO (250 ng/mL) for 5 min at 4°C, washed and shifted to 37°C in medium containing or not Ca²⁺ for increasing periods of time. Cell extracts are then incubated with GFP-Trap for pulldown of total GFP-SLO and analyzed by western blot. A) GFP-SLO immunoblot at increasing periods after cell permeabilization in the presence (repair conditions) or absence (no repair conditions) of Ca²⁺. The amount of cell-associated GFP-SLO decreases rapidly between 5 and 30 min in cells permeabilized in the presence but not in the absence of Ca²⁺. B) Densitometric quantification of GFP-SLO after various time-points in the presence or absence of Ca²⁺, expressed as percentage of the amount detected at t = 5 min. Results are expressed as the mean ± SEM of eight independent experiments. **p < 0.01, ***p < 0.001, unpaired Student’s t-test. C–E) Cells were incubated with SLO for 5 min at 4°C followed by a 10-min incubation at 37°C with DQ-BSA and chased for increasing periods of time. C) Representative images of cells treated or not with 250 ng/mL SLO and incubated with DQ-BSA, followed by a chase for 2 h in the presence or absence of BafA. SLO treatment leads to a marked enhancement in DQ-BSA fluorescence, reflecting the increased number of acidified late endocytic compartments. Blocking acidification with BafA abolishes DQ-BSA fluorescence. DQ-BSA fluorescence is depicted in white and DAPI-stained nuclei in gray. Bar = 5 μm. D) Quantification of DQ-BSA fluorescence intensity in control and SLO-treated cells over time. Exposure to SLO progressively increases the fluorescence intensity of DQ-BSA over a 30-, 60- and 120-min chase period, reflecting acidification of the endocytic compartments induced by the toxin. The values reflect the mean fluorescence intensity of five independent microscopic fields per sample. E) SLO permeabilization causes a dose-dependent increase in the fluorescence intensity of endocytosed DQ-BSA after a 2-h chase. Results in (E and F) are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, unpaired Student’s t-test comparing all samples to the untreated control. F) Control cells or cells treated with concanamycin A/leupeptin or MG132 were incubated with GFP-SLO (250 ng/mL) for 5 min at 4°C, shifted to 37°C in medium containing Ca²⁺ for 5 or 30 min and processed for GFP pulldown and immunoblot. Lysosomal, but not proteasome, inhibitors blocked GFP-SLO degradation.