Figure 6: Lowering PI4P levels with PAO rescues excessive retromer tubulation and cholesterol accumulation in LE/LYS in Arf6 KO cells.

(a) Normalized integrated densities of PI4P staining (see d) of Arf6 KO cells treated with DMSO (100±9%,±indicates s.e.m., n=22 cells, 3 experiments) or PAO (79±4%, n=29 cells, three experiments). *P<0.05 in t-test with Welch’s correction. (b) Bar diagram showing PI4P levels in DMSO- and PAO-treated Arf6 KO cells. Measurements were made by anionic exchange HPLC with suppressed conductivity detection, expressed in molar percentage of measured lipids and normalized to DMSO-treated Arf6 KO levels. PI4P levels were decreased in PAO-(63±5%, ± indicates s.e.m., n=4) compared with DMSO-treated controls (100±1%, n=4). ***P<0.001 in Student’s t-test. (c) Kinetic following of GFP-SNX6 tubulation in Arf6 KO MEFs treated with DMSO or PAO by spinning-disk live imaging. Top panel, representative images of Arf6 KO cells after different times of PAO treatment. Arrows indicate tubules. Scale bar, 5 and 1 μm (zoom-ins). Lower panel, quantification of the number of GFP-SNX6 tubules after DMSO (black, n=9 cells, four experiments) or PAO treatment (red, n=9 cells, four experiments), normalized to the number of tubules before the treatment. *P<0.05 and ***P<0.001 in two-way ANOVA with Bonferroni post-tests, respectively. (d) Left, representative immunostaining for PI4P (magenta) of Arf6 KO cells treated with DMSO or PAO and labelled with filipin (blue). Scale bar, 5 μm. Middle, quantification of the absolute number and relative distribution of filipin puncta in DMSO-(37±3 puncta/cell,±indicates s.e.m., n=37 cells, 4 experiments) or PAO-(30±3 puncta per cell, n=29 cells, four experiments) treated Arf6 KO cells. NS denotes P>0.05 in Student’s t-test. **P<0.01 in χ²-test. Right, size of filipin puncta in DMSO-(0.53±0.01 μm²,±indicates s.e.m., 1,352 puncta, 37 cells) or PAO-(0.50±0.01 μm², 876 puncta, 29 cells) treated Arf6 KO cells. **P<0.01 in Mann–Whitney test. NS, not significant.