Figure 3: Retromer function is impaired in Arf6 KO MEFs. | Nature Communications

Figure 3: Retromer function is impaired in Arf6 KO MEFs.

From: Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Figure 3

(a) Representative maximum intensity projections of Arf6 WT and KO cells immunostained for CI-M6PR (magenta) and Vps35 (green). Scale bar, 5 μm. (b) Western blot analysis of Vps35 levels in Arf6 WT (100±5%, n=5) and KO cells (113±5%, n=5), P>0.05 in Student’s t-test. Tubulin was used as an equal loading marker. (c) Representative confocal images of Arf6 WT and KO cells immunostained for EEA1 (magenta) and Vps35 (green). Scale bar, 5 μm. (d) Levels of Vps35/EEA1 co-localization in Arf6 WT (10±1%, n=26 cells) and KO cells (15±1%, n=30 cells). **P<0.01 in t-test with Welch’s correction. (e) Top left panel, number of Vps35 puncta in Arf6 WT (116±8 puncta per cell, n=28 cells) and KO cells (126±6 puncta per cell, n=28 cells). ns stands for P>0.05 in Student’s t-test. Top right, size of Vps35 puncta in WT (0.53±0.01 μm2, 3,241 puncta) and KO cells (0.59±0.01 μm2, 3,529 puncta). ** stands for P<0.01 in Mann–Whitney test. Bottom left, number of EEA1 puncta in WT (235±12 puncta per cell, n=28 cells) and KO cells (232±13 puncta per cell, n=23 cells). Bottom right, size of EEA1 puncta in WT (0.40±0.01 μm2, 6,571 puncta) and KO cells (0.39±0.01 μm2, 5,334 puncta). ns stands for P>0.05 in Student’s t-test (left) and Mann–Whitney test (right). (f) Maximum intensity projections of 3D SIM z-stacks of Arf6 WT and KO cells immunostained for EEA1 (magenta) and Vps35 (green). Scale bar, 5 μm. (g) Spinning-disk confocal imaging of Arf6 WT and KO MEFs expressing GFP-SNX6. Top panel, representative images. Scale bar, 5 and 1 μm (insets). Arrows indicate tubules. Lower panel, quantification of GFP-SNX6 tubules. Left, number of GFP-SNX6 tubules in Arf6 WT (3±0.3 tubules per cell, n=21 cells) and KO cells (4.5±0.6 tubules per cell, n=22 cells). *P<0.05 in t-test with Welch’s correction. Center, frequency distribution of GFP-SNX6 tubules length in Arf6 WT (2.6±0.2 μm, n=63 tubules) and KO cells (3.2±0.2 μm, n=97 tubules). ***P<0.001 in χ2-test. Right, persistence of GFP-SNX6 tubules in Arf6 WT (11.6±1.8 s, n=62 tubules) and KO cells (24.1±2.9 s, n=97 tubules). ***P<0.001 in t-test with Welch’s correction. All values are given as mean±s.e.m.

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