Figure 1.
SNX10 localizes to early and late endocytic compartments. (A) Graphical view of the SNX10 isoforms annotated in UniProt. The PX domain is represented in green, and the numbers indicate the number of amino acids. The arrows indicate the position of the natural variants (SNPs) linked to ARO (Y32S, R51P, and R51Q). (B) The figure displays the predicted protein structure of SNX10 generated using AlphaFold, showcasing its three-dimensional conformation. (C) Confocal imaging of U2OS cell lines stably expressing doxycycline-inducible SNX10-EGFP WT or the indicated ARO-linked mutants. Nuclei were stained with Hoechst. Scale bar: 20 µm. (D) Representative immunoblot showing the expression levels of SNX10-EGFP and the indicated ARO mutants. The membrane was blotted using an anti-GFP antibody and using actin as a loading control. (E) Representative immunofluorescence images of U2OS cells stably expressing SNX10-EGFP WT or the Y32S mutant (green) immunostained with anti-EEA1 (magenta) after treating cells with 5 µM VPS34-IN1 for 2 h. Nuclei were stained with Hoechst. Scale bar: 10 µm. Insets: 9.35 × 9.35 µm. (F) Representative image of U2OS cells stably expressing SNX10-EGFP and immunostained with anti-CD63 (magenta) and anti-EEA1 (yellow) antibodies. Images were taken with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 5.12 × 5.12 µm. (G) Quantification of F represented as the percentage of SNX10 structures that are either CD63- or EEA1-positive. Data are mean ± SEM with individual data points corresponding to a single field of view (n > 300 cells, four experiments). The significance was assessed by unpaired t test. Data distribution was assumed to be normal, but this was not formally tested. (H) Representative immunofluorescence images of U2OS cells stably expressing SNX10-EGFP WT or the Y32S mutant (green) immunostained with anti-LAMP1. Scale bar: 10 µm. Insets: 10.43 × 10.43 µm. (I) U2OS SNX10-EGFP cells fixed for CLEM analysis. The area analyzed and shown in (ii) is indicated with a square in the confocal image (i). (iii) Shows the transmission EM and (iv) the z-slide from the tomogram from the white dotted area shown in (ii). The white arrows indicate clathrin-coated vesicles. (v) Green: endosomes; red: lipid droplets; yellow: vesicles; and pink: clathrin-coated vesicles. Scale bars: 10 μm (i and ii), 1 μm (iii and iv). SNPs; single nucleotide polymorphisms. Source data are available for this figure: SourceData F1.

SNX10 localizes to early and late endocytic compartments. (A) Graphical view of the SNX10 isoforms annotated in UniProt. The PX domain is represented in green, and the numbers indicate the number of amino acids. The arrows indicate the position of the natural variants (SNPs) linked to ARO (Y32S, R51P, and R51Q). (B) The figure displays the predicted protein structure of SNX10 generated using AlphaFold, showcasing its three-dimensional conformation. (C) Confocal imaging of U2OS cell lines stably expressing doxycycline-inducible SNX10-EGFP WT or the indicated ARO-linked mutants. Nuclei were stained with Hoechst. Scale bar: 20 µm. (D) Representative immunoblot showing the expression levels of SNX10-EGFP and the indicated ARO mutants. The membrane was blotted using an anti-GFP antibody and using actin as a loading control. (E) Representative immunofluorescence images of U2OS cells stably expressing SNX10-EGFP WT or the Y32S mutant (green) immunostained with anti-EEA1 (magenta) after treating cells with 5 µM VPS34-IN1 for 2 h. Nuclei were stained with Hoechst. Scale bar: 10 µm. Insets: 9.35 × 9.35 µm. (F) Representative image of U2OS cells stably expressing SNX10-EGFP and immunostained with anti-CD63 (magenta) and anti-EEA1 (yellow) antibodies. Images were taken with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 5.12 × 5.12 µm. (G) Quantification of F represented as the percentage of SNX10 structures that are either CD63- or EEA1-positive. Data are mean ± SEM with individual data points corresponding to a single field of view (n > 300 cells, four experiments). The significance was assessed by unpaired t test. Data distribution was assumed to be normal, but this was not formally tested. (H) Representative immunofluorescence images of U2OS cells stably expressing SNX10-EGFP WT or the Y32S mutant (green) immunostained with anti-LAMP1. Scale bar: 10 µm. Insets: 10.43 × 10.43 µm. (I) U2OS SNX10-EGFP cells fixed for CLEM analysis. The area analyzed and shown in (ii) is indicated with a square in the confocal image (i). (iii) Shows the transmission EM and (iv) the z-slide from the tomogram from the white dotted area shown in (ii). The white arrows indicate clathrin-coated vesicles. (v) Green: endosomes; red: lipid droplets; yellow: vesicles; and pink: clathrin-coated vesicles. Scale bars: 10 μm (i and ii), 1 μm (iii and iv). SNPs; single nucleotide polymorphisms. Source data are available for this figure: SourceData F1.

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