FIGURE 7:

Disruption of HOPS decreases NPC2 entering endosome-lysosome system. WT and VPS39-KO cells were transfected with the plasmids encoding NPC2-mCherry and fixed for immunostaining with antibodies to LAMP2 and EEA1 (A), or Rab5 (C), or Rab7a (E) at 24 h posttransfection. Scale bars, 5 μm. Parts of a cell are shown at high magnification, and Rab7a-positive and -negative NPC2 vesicles are pointed by arrows in panels 1 and 2, respectively. Scale bar, 1 μm (E) Cells were analyzed for the colocalization between LAMP2 and EEA1 (B, n = 19 and 24), LAMP2 and Rab5 (D, n = 23 and 24), and LAMP2 and Rab7a (F, n = 30, and 32). (G) Cells in E were further analyzed for the quantities of LAMP2-positive, LAMP2-negative/Rab7a-positive, and LAMP2-negative/Rab7a-negative NPC2 vesicles in WT (n = 8) and VPS39-KO (n = 7) cells. The results were shown as the percentage of each group NPC2 vesicles out of the total number of NPC2 vesicles; p values were determined by Student’s t test. ****p < 0.0001, n.s., not significant (vs. WT).