Figure 4.

Acetate induces LMP, analyzed by lysosomal pH alterations and CatD release to the cytosol. (a) HCT-15 and RKO cells were incubated with acetate (70 mM, 100 mM and 140 mM, for HCT-15 cells; 110 mM, 140 mM and 220 mM, for RKO cells) for 48 h or with fresh complete medium or etoposide (50 μM) as a negative and positive control, respectively. LMP was detected by AO staining and visualization by fluorescence microscopy. Representative images ( × 400) are shown. (b) Representative images of monoparametric histograms of green fluorescence (FL3 area (log)) in HCT-15 cells treated as in (a). (c) Percentage of HCT-15 and RKO cells displaying LMP quantified by flow cytometry analysis of AO staining after exposure to acetate as described in (a). Values represent mean±S.E.M. of three independent experiments. *P≤0.05; **P≤0.01 compared with negative control cells. (d) Effect of acetate on the expression and release of cathepsin D to the cytosol in HCT-15 and RKO cells, comparing whole-cell lysates (total) and cytosolic fractions (cyto). Cells were treated with IC₅₀ and 2 × IC₅₀ acetate concentrations (respectively, 70 mM and 140 mM for HCT-15 cells and 110 mM and 220 mM for RKO cells) for 48 h or with fresh complete medium or etoposide (50 μM) as a negative and positive control, respectively. Actin was used as a loading control