Figure S4.
SNX13 depletion redistributes cholesterol to the cell surface in the absence of NPC1 function.(A) U2OS cells were treated with SNX13 siRNA for 72 h. After 24 h, cells were transfected with plasmid encoding siRNA-resistant HA-tagged WT-SNX13. 16 h before fixation, cells were treated with U18666A for 16 h and then labeled with GST-PFO detected using anti-GST primary antibodies. Scale bar, 10 µm. Insets show enlargements of the boxed areas from cells rescued with siRNA-resistant SNX13-HA (box 1) or untransfected cells (box 2); scale bars, 2 µm. (B and C) Quantitation of PFO*-positive vesicles and vesicular PFO fluorescence intensity determined by CellProfiler. Colored dots reflect means from independent experiments; 64 rescued cells from a total of >300 cells were analyzed. Significance was determined by unpaired t test; *, P < 0.05. (D) Immunofluorescence microscopy of HeLa cells treated with U18666A for 16 h (left column images) or left untreated (right column images) and labeled in the absence of detergent permeabilization with GST-PFO detected using anti-GST primary antibodies 72 h after transfection with the indicated siRNAs. (E) Quantitation of PFO staining as a function of PM cell area in U18666A-treated (top plot) and untreated (bottom plot) cells determined using CellProfiler. Colored dots reflect means from independent experiments; >830 cells analyzed in each condition. Significance was determined by unpaired t test; **, P < 0.01. (F) Immunofluorescence microscopy of NPC1-knockout (KO) HeLa cells stained in the absence of detergent permeabilization with GST-PFO detected by anti-GST primary antibodies. (G and H) U2OS cells treated with U18666A for 16 h (G) or left untreated (H), labeled with anti-BMP antibodies, and imaged by confocal microscopy. Scale bars, 20 µm. Actual P values: (B) Untransfected + U18 versus + WT-SNX13 + U18, P = 0.023; (C) untransfected+U18 versus +WT-SNX13+U18, P = 0.047; (E) top: Ctrl siRNA+U18 versus SNX13 siRNA+U18, P = 0.0017; (E) bottom: Ctrl siRNA versus SNX13 siRNA, P = 0.377. AU, arbitrary unit.

SNX13 depletion redistributes cholesterol to the cell surface in the absence of NPC1 function. (A) U2OS cells were treated with SNX13 siRNA for 72 h. After 24 h, cells were transfected with plasmid encoding siRNA-resistant HA-tagged WT-SNX13. 16 h before fixation, cells were treated with U18666A for 16 h and then labeled with GST-PFO detected using anti-GST primary antibodies. Scale bar, 10 µm. Insets show enlargements of the boxed areas from cells rescued with siRNA-resistant SNX13-HA (box 1) or untransfected cells (box 2); scale bars, 2 µm. (B and C) Quantitation of PFO*-positive vesicles and vesicular PFO fluorescence intensity determined by CellProfiler. Colored dots reflect means from independent experiments; 64 rescued cells from a total of >300 cells were analyzed. Significance was determined by unpaired t test; *, P < 0.05. (D) Immunofluorescence microscopy of HeLa cells treated with U18666A for 16 h (left column images) or left untreated (right column images) and labeled in the absence of detergent permeabilization with GST-PFO detected using anti-GST primary antibodies 72 h after transfection with the indicated siRNAs. (E) Quantitation of PFO staining as a function of PM cell area in U18666A-treated (top plot) and untreated (bottom plot) cells determined using CellProfiler. Colored dots reflect means from independent experiments; >830 cells analyzed in each condition. Significance was determined by unpaired t test; **, P < 0.01. (F) Immunofluorescence microscopy of NPC1-knockout (KO) HeLa cells stained in the absence of detergent permeabilization with GST-PFO detected by anti-GST primary antibodies. (G and H) U2OS cells treated with U18666A for 16 h (G) or left untreated (H), labeled with anti-BMP antibodies, and imaged by confocal microscopy. Scale bars, 20 µm. Actual P values: (B) Untransfected + U18 versus + WT-SNX13 + U18, P = 0.023; (C) untransfected+U18 versus +WT-SNX13+U18, P = 0.047; (E) top: Ctrl siRNA+U18 versus SNX13 siRNA+U18, P = 0.0017; (E) bottom: Ctrl siRNA versus SNX13 siRNA, P = 0.377. AU, arbitrary unit.

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