Figure 6.

CQ does not impair endocytosis and endo-lysosomal trafficking of BSA but causes vacuolization of lysosomes. (A) U2OS cells were exposed to 100 µM CQ or 100 nM BafA₁ for 2 h or left untreated, before being incubated with 375 nM BSA-TRITC (BSA-T) for 30 min. The cells were then washed and further incubated in the same medium with 100 µM CQ or 100 nM BafA₁ and without BSA-TRITC for 90 min. Finally, cells were processed for immunofluorescence microscopy and stained with anti-LAMP2 antibodies. The white arrow indicates large BSA-TRITC-positive LAMP2 puncta. (B) Quantification of the colocalization between BSA-TRITC (BSA-T) and LAMP2 puncta in the experiment shown in panel A. (C) Determination of the average size of the BSA-TRITC (BSA-T)-, LAMP2- and BSA-TRITC/LAMP2 (coloc)-positive puncta (arbitrary units) in the experiment shown in panel A. (D) U2OS cells were incubated with 375 nM BSA-TRITC (BSA-T) for 30 min, washed and further incubated in medium without BSA-TRITC for 90 min before being exposed to 100 µM CQ or 100 nM BafA₁ for 5 h, or left untreated. Cells were subsequently prepared for immunofluorescence microscopy and labeled with anti-LAMP2 antibodies. The white arrow indicates large BSA-TRITC-positive LAMP2 puncta. (E) Quantification of the colocalization between BSA-TRITC (BSA-T) and LAMP2 puncta in the experiment shown in panel D. (F) Determination of the average size of the BSA-TRITC (BSA-T)-, LAMP2- and BSA-TRITC/LAMP2 (coloc)-positive puncta (arbitrary units) in the experiment shown in panel D. All images were acquired using the DeltaVision microscope. Data in panels C and F are presented relative to the control (folds). Error bars represent SD of 3 independent experiments. Symbols * and ** indicate significant differences of p < 0.05 and p < 0.01, respectively. Scale bars: 10 µm.