FIGURE 5.

ATP6V1A is required for basal lysosomal function. (a) OE‐NC and OE‐ATP6V1A cells were cultured under hypoxia for 48 h, and stained with Lysotracker Red. Lysotracker⁺ dots were quantified. Scale bar, 20 μm. (b) Lysosomal pH was determined using Lysosensor™ Yellow/Blue DND‐160 after cells were exposed to hypoxia for 48 h. (c) Confocal microscopy analysis of LAMP1 in OE‐NC and OE‐ATP6V1A cells to assess the size of lysosomes. Scale bar, 25 μm. The right graph shows the distribution of the lysosomal diameter (n > 70 per group). (d) Confocal co‐localization analysis of LAMP1 and Rab7 in OE‐NC and OE‐ATP6V1A cells. Scale bar, 25 μm. The ratio of co‐localization of LAMP1 and Rab7 was quantified (n > 10 per group). (e) Electron microscopy images showing MVBs in OE‐NC and OE‐ATP6V1A cells. Scale bar, 500 nm. Right graph: quantification of ILV number per MVB. Each dot represents the number of ILVs per MVB (n > 10 per group). (f) The EVs were collected from equal numbers of OE‐NC/OE‐ATP6V1A cells and concentration of EV protein was measured using BCA assay. (g) The size and quantity of EVs from OE‐NC/ OE‐ATP6V1A cells were measured using NTA. EVs were collected from 20 × 10⁶ cells for each group. Statistical analyses were performed using t‐test. ** P‐value < 0.01, *** P‐value < 0.001, **** P‐value < 0.0001. Data are presented as the mean ± S.D.