SNX13 overexpression induces ER–EL membrane contacts . (A) U2OS cells were transfected with the GFP-tagged ER control probe HNeu (GFP-ER) or SNX13-GFP. 24 h after transfection, ELs were loaded with TRITC-labeled dextran for 4 h at 37°C. Live images at left correspond to cells expressing GFP-ER (top) or SNX13-GFP (bottom) under isotonic conditions (left column) or hypotonic media (right column). Insets to the right of isotonic conditions show control (GFP-ER) or SNX13-labeled ER domains in proximity to ELs (yellow arrowheads). Consecutive image frames at right correspond to time-lapse recording of boxed areas (insets 1 and 2) from both control (top) and SNX13-expressing cells (bottom) under hypotonic conditions. Scale bars, 10 µm; enlarged insets, 1 µm. (B–D) Quantitation of persistence of ER–EL contacts (B), average number of ER–EL contacts per frame (C), and average ER–EL contact length per frame (D). Colored dots reflect means from independent experiments; 15 cells analyzed in each condition. Significance was determined by unpaired t test; *, P < 0.05. Actual P values: (B) +GFP-ER versus +SNX13-GFP, P = 0.02; (C) +GFP-ER versus +SNX13-GFP, P = 0.017; (D) +GFP-ER versus +SNX13-GFP, P = 0.035. (E) Model depicting a possible role for SNX13 in endosomal cholesterol egress regulation.