Figure 2: NPC2 mistrafficking causes cholesterol redistribution in Arf6 KO MEFs.
(a) Left panel, representative confocal images of Arf6 WT and KO cells immunostained for endogenous LAMP1 (magenta) and NPC2 (green). Scale bar, 5 μm. Right panel, NPC2/LAMP1 co-localization levels in Arf6 WT (23±1%,±indicates s.e.m., n=36 cells from three experiments) and Arf6 KO cells (16±1%, n=24 cells from three experiments). ***P<0.001 in Student’s t-test. (b) Western blot analysis of endogenous NPC2 levels in Arf6 WT and KO cells. Tubulin was used as an equal loading marker. NPC2 protein levels were similar (P>0.05 in Student’s t-test) in Arf6 WT (100±6%,±indicates s.e.m., n=3) and KO cells (110±26%, n=3). (c) NPC2-Alexa488 (green) or vehicle was added to the culture media of Arf6 WT and KO cells for 24 h. Cells were then washed, fixed and stained for endogenous LAMP1 (magenta) and cholesterol (with filipin, blue). Left panel, representative confocal images. Scale bar, 5 μm. Inset shows that exogenous NPC2 reaches the LAMP1 compartment. Center and right panel, quantification of filipin puncta. Center, number of filipin puncta in vehicle-treated Arf6 WT (16±2 puncta per cell,±indicates s.e.m., n=34 cells, three experiments), vehicle-treated KO (24±3 puncta per cell, n=35 cells, three experiments), NPC2-treated Arf6 WT (18±2 puncta per cell, n=36 cells, three experiments) and NPC2-treated KO cells (18±5 puncta per cell, n=28 cells, three experiments). NS and * stand for P>0.05 and P<0.05, respectively, in t-test with Welch’s correction. Right, scatter dot blot of the size of filipin puncta of vehicle-treated Arf6 WT (0.41±0.01 μm2,±indicates s.e.m., 540 puncta from 34 cells), vehicle-treated Arf6 KO (0.48±0.01 μm2, 843 puncta from 35 cells), NPC2-treated Arf6 WT (0.46±0.01 μm2, 663 puncta from 36 cells) and NPC2-treated Arf6 KO cells (0.45±0.01 μm2, 496 puncta from 28 cells). NS denote P>0.05, *P<0.05 and ***P<0.001 in Mann–Whitney test, respectively. NS, not significant.
