Figure 4.

Expression of ATP6V1B2 and ATP6V1C1 mutants is associated with increased acidification of lysosomes

(A) Patients’ and control cells were incubated with 2 μM LysoSensor Yellow/Blue DND-160 (Thermo Fisher Scientific) and observed on a Zeiss LSM 980 confocal microscope. Images were acquired using a 63× oil objective, laser and filter settings were adjusted according to the fluorescence excitation and emission requirements of the reagent. Scale bar, 5 μm.

(B) Evaluation of lysosomal pH values in patient-derived fibroblasts. The pH calibration curve performed using WT1 cells, and obtained by plotting the fluorescence intensity 450/540 ratios as a function of pH, was fitted with linear regression using GraphPad Prism5 software (left). Data are means ± SEM from >50 cells analyzed for each pH value. Colored filled circled represent the experimentally measured ratios converted into absolute pH values by interpolation in the pH calibration curve. Representative images of fibroblasts incubated with LysoSensor Yellow/Blue dextran and visualized in live at 450 nm (top) and 540 nm (bottom) emission wavelengths (right). The cell contours recognition was performed in bright field images and used to effectively quantify fluorescence intensity. Scale bar, 20 μm. All panels are identical in scale.