Figure 1: Cholesterol is redistributed to late endosomes/lysosomes in Arf6 KO MEFs. | Nature Communications

Figure 1: Cholesterol is redistributed to late endosomes/lysosomes in Arf6 KO MEFs.

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

Figure 1

(a) Western blot analysis of endogenous Arf6 levels in Arf6Flox/Flox; Cre-ER MEFs treated with DMSO (Arf6 WT) or tamoxifen (Arf6 KO). Tubulin was used as an equal loading marker. (b) Cholesterol levels measured by LC–MS were similar in Arf6 WT (19.1±0.7 molar % of total lipidome,±indicates s.e.m., n=11) and KO cells (18.5±0.5 molar % of total lipidome, n=12). NS denotes P>0.05 in Student’s t-test. (c) Representative confocal images of Arf6 WT and KO MEFs stained with filipin. Scale bar, 5 μm. (d) Normalized integrated densities of filipin staining were similar in Arf6 WT (100±8%,±indicates s.e.m., n=32 cells from three experiments) and Arf6 KO cells (98±8%, n=42 cells from four experiments). NS denotes P>0.05 in Student’s t-test. (e) Quantification of filipin puncta in Arf6 WT and KO cells. Left panel, number of puncta per cell in Arf6 WT (13±2 puncta per cell,±indicates s.e.m., n=39 cells from 3 experiments) and Arf6 KO cells (26±3 puncta per cell, n=29 cells from 3 experiments). **P<0.01 in Student’s t-test. Middle panel, frequency distribution of the number of filipin puncta per cell. ***P<0.001 in χ2-test. Right panel, scatter dot blot of the size of filipin puncta in Arf6 WT (0.42±0.01 μm2,±indicates s.e.m., 498 puncta from 39 cells) and KO cells (0.47±0.01 μm2, 749 puncta from 29 cells). *P<0.05 in Mann–Whitney test. (f) Representative immunostainings for EEA1, Rab7 or LAMP1 (magenta) of Arf6 WT or KO cells labelled with filipin (green). Scale bar, 5 μm. NS, not significant.

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